Forms of a multicyclic compound

ABSTRACT

The present invention provides alternative forms of Compound I, 
                         
processes to reproducibly make them and methods of treating patients using them.

FIELD OF THE INVENTION

The present invention relates to compositions which contain novel formsof a multicyclic compound (hereinafter referred to as Compound I),processes to reproducibly make them and pharmaceutical compositionscomprising Compound I.

BACKGROUND OF THE INVENTION

Active pharmaceutical ingredients (APIs) can be prepared in a variety ofdifferent forms, for example, chemical derivatives, solvates, hydrates,co-crystals, or salts. APIs may also be amorphous, may have differentcrystalline polymorphs, or may exist in different solvation or hydrationstates. By varying the form of an API, it is possible to vary thephysical properties thereof. For instance, crystalline polymorphstypically have different solubilities such that a more thermodynamicallystable polymorph is less soluble than a less thermodynamically stablepolymorph. Polymorphs can also differ in properties such as stability,bioavailability, morphology, vapor pressure, density, color, andcompressibility. Accordingly, variation of the crystalline state of anAPI is one of many ways in which to modulate the physical andpharmacological properties thereof.

Poly(ADP-ribose) polymerase (PARP, also called poly(ADP-ribose)synthetase, or PARS) is a nuclear enzyme which catalyzes the synthesisof poly(ADP-ribose) chains from NAD⁺ in response to single-stranded DNAbreaks as part of the DNA repair process (de Murcia, G; de Murcia, J. M.Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem.Sci. 1994, 19,172-176; Alvarez-Gonzalez, R.; Pacheco-Rodriguez, G.;Mendoza-Alvarez, H. Enzymology of ADP-ribose polymer synthesis. Mol.Cell. Biochem. 1994, 138, 33). It has been hypothesized that smallmolecule inhibitors of PARP may play a potential role in the therapeutictreatment of neurodegenerative disorders, cancers, and other PARP andkinase-related diseases.

A specific PARP inhibitor compound, having the chemical designation4,5,6,7-tetrahydro-11-methoxy-2-[(4-methyl-1-piperazinyl)methyl]-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)-dionemay have utility in the treatment of breast and ovarian tumors and inconjunction with chemotherapy or radiotherapy for the treatment of otherdrug-resistant cancers. This compound is represented by the followingformula (I):

and is referred to hereinafter as “Compound I”. U.S. Pat. No. 7,122,679and U.S. 2006/0276497 describe Compound I and utility thereof.

Different forms of Compound I can have different melting points,solubilities or rates of dissolution; these physical properties, eitheralone or in combination, can affect, for example, bioavailability. Inlight of the potential benefits of alternative forms of APIs, a needexists to identify and prepare alternative forms of Compound I.

SUMMARY OF THE INVENTION

Various forms of Compound I are described, as well as methods for theirpreparation. Specifically, two polymorphs of anhydrous crystalline forms(Forms A₀ and B₀), three polymorphs of crystalline monohydrate forms(HA₀, HC₀ and HD₀) and nine solvates (S2₀, S3₀, S4₀, S5₀, S6₀, S7₀, S9₀,S10₀ and S12₀) are described herein. Pharmaceutical compositionscomprising one or more of these forms are also described, as well aspharmaceutical compositions further comprising an amorphous form ofCompound I (A_(s)). Pharmaceutical compositions comprising one or moreof these forms are also described, as are methods of treatment utilizingsuch compositions.

The pharmaceutical compositions of the present invention may be used ina variety of ways, including but not limited to the enhancement of theanti-tumor activity of radiation or DNA-damaging chemotherapeutic agents(Griffin, R. J.; Curtin, N. J.; Newell, D. R.; Golding, B. T.; Durkacz.B. W.; Calvert, A. H. The role of inhibitors of poly(ADP-ribose)polymerase as resistance-modifying agents in cancer therapy. Biochemie1995, 77, 408).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an X-ray Powder Diffractogram (XRPD) of Form A₀.

FIG. 2 is an X-ray Powder Diffractogram (XRPD) of Form B₀.

FIG. 3 is an X-ray Powder Diffractogram (XRPD) of Form HA₀.

FIG. 4 is an X-ray Powder Diffractogram (XRPD) of Form HC₀.

FIG. 5 is an X-ray Powder Diffractogram (XRPD) of Form HD₀.

FIG. 6 is an X-ray Powder Diffractogram (XRPD) of Form S2₀.

FIG. 7 is an X-ray Powder Diffractogram (XRPD) of Form S3₀.

FIG. 8 is an X-ray Powder Diffractogram (XRPD) of Form S4₀.

FIG. 9 is an X-ray Powder Diffractogram (XRPD) of Form S5₀.

FIG. 10 is an X-ray Powder Diffractogram (XRPD) of Form S6₀.

FIG. 11 is an X-ray Powder Diffractogram (XRPD) of Form S7₀.

FIG. 12 is an X-ray Powder Diffractogram (XRPD) of Form S9₀.

FIG. 13 is an X-ray Powder Diffractogram (XRPD) of Form S10₀.

FIG. 14 is an X-ray Powder Diffractogram (XRPD) of Form S12₀.

FIG. 15 is a Variable Temperature X-ray Powder Diffractogram (VT-XRPD)of Form A₀.

FIG. 16 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form A₀.

FIG. 17 is a Dynamic Vapor Sorption (DVS) regular isotherm plot of FormA₀.

FIG. 18 depicts X-ray Powder Diffractograms (XRPD) of Form A₀ before andafter Dynamic Vapor Sorption (DVS) analysis.

FIG. 19 is a Dynamic Vapor Sorption (DVS) irregular isotherm plot ofForm A₀.

FIG. 20 is a Fourier Transform Infrared (FTIR) spectrum of Form A₀.

FIG. 21 is a Raman Spectrum of Form A₀.

FIG. 22 is a Variable Temperature X-ray Powder Diffractogram (VT-XRPD)of Form B₀.

FIG. 23 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form B₀.

FIG. 24 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form HA₀.

FIG. 25 is a Dynamic Vapor Sorption (DVS) isotherm plot of Form HA₀.

FIG. 26 is a Fourier Transform Infrared (FTIR) spectrum of Form HA₀.

FIG. 27 is a Raman spectrum of Form HA₀.

FIG. 28 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form HC₀.

FIG. 29 is a Dynamic Vapor Sorption (DVS) regular isotherm plot of FormHC₀.

FIG. 30 depicts X-ray Powder Diffractograms (XRPD) of Form HC₀ beforeand after Dynamic Vapor Sorption (DVS) analysis.

FIG. 31 is a Dynamic Vapor Sorption (DVS) irregular isotherm plot ofForm HC₀.

FIG. 32 is a Fourier Transform Infrared (FTIR) spectrum of Form HC₀.

FIG. 33 is a Raman spectrum of Form HC₀.

FIG. 34 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form HD₀.

FIG. 35 is a Dynamic Vapor Sorption (DVS) regular isotherm plot of FormHD₀.

FIG. 36 depicts X-ray Powder Diffractograms (XRPD) of Form HD₀ beforeand after Dynamic Vapor Sorption (DVS) analysis.

FIG. 37 is a Dynamic Vapor Sorption (DVS) irregular isotherm plot ofForm HD₀.

FIG. 38 is a Fourier Transform Infrared (FTIR) spectrum of Form HD₀.

FIG. 39 is a Raman spectrum of Form HD₀.

FIG. 40 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S2₀.

FIG. 41 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S3₀.

FIG. 42 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S4₀.

FIG. 43 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S5₀.

FIG. 44 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S6₀.

FIG. 45 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S7₀.

FIG. 46 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S9₀.

FIG. 47 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S10₀.

FIG. 48 is a Differential Scanning calorimetry (DSC) Thermogram andThermo-Gravimetric Analysis (TGA) Thermogram overlay of Form S12₀.

FIG. 49 depicts an overlay of X-ray Powder Diffractogram (XRPD) patternsof Form A₀ after grinding.

FIG. 50 depicts an overlay of X-ray Powder Diffractogram (XRPD) patternsof Form HC₀ and HD₀ after grinding for 15 minutes.

FIG. 51 is a Differential Scanning calorimetry (DSC) Thermogram of FormHC₀ and HD₀ after grinding for 15 minutes.

DETAILED DESCRIPTION OF THE INVENTION

The existence has now been found of a number of forms of Compound I. Thepreparation and description of these forms is described herein. Spectraldata relating to these forms are shown in FIGS. 1-51.

More specifically, the existence has been found of a number of differentphysical forms of Compound I. Two polymorphs of anhydrous crystallineforms of Compound I (Forms A₀ and B₀), and three polymorphs ofcrystalline monohydrate forms (HA₀, HC₀ and HD₀) have been discovered.The letters A and B were assigned for these anhydrous forms andhydrates, with the leading H specifically denoting the hydrate forms.The subscript ‘0’ was further assigned to identify the free base forms.In addition, nine solvates of Compound I (S2₀, S3₀, S4₀, S5₀, S6₀, S7₀,S9₀, S10₀ and S12₀) are described herein. Pharmaceutical compositionscomprising one or more of these forms are also described, as well aspharmaceutical compositions further comprising an amorphous form ofCompound I (A_(S)).

Representative XRPD peaks for Form A₀ are listed in the followingTable 1. The X-Ray diffraction pattern characteristic of Form A₀ isshown in FIG. 1.

TABLE 1 Form A₀ XRPD peaks Angle Intensity Peak No. [°2 Theta] d-spacing[Angstrom] [%] 1 4.32 20.42 100 2 6.07 14.55 99 3 8.55 10.34 79 4 9.549.26 44 5 12.07 7.33 69 6 12.78 6.92 31 7 13.48 6.56 11 8 15.37 5.76 809 18.09 4.90 40 10 19.09 4.65 17 11 23.77 3.74 5 12 24.16 3.68 7 1324.54 3.62 6 14 27.41 3.25 7

Representative XRPD peaks for Form B₀ are listed in the following Table2. The X-Ray diffraction pattern characteristic of Form B₀ is shown inFIG. 2.

TABLE 2 Form B₀ XRPD peaks Angle Intensity Peak No. [°2 Theta] d-spacing[Angstrom] [%] 1 7.16 12.33 36 2 7.89 11.20 100 3 10.55 8.38 6 4 10.778.21 22 5 15.81 5.60 7 6 16.54 5.35 28 7 18.53 4.78 6 8 19.27 4.60 9 921.20 4.19 18 10 24.04 3.70 6 11 24.61 3.62 17 12 24.65 3.61 16

Representative XRPD peaks for Form HA₀ are listed in the following Table3. The X-Ray diffraction pattern characteristic of Form HA₀ is shown inFIG. 3.

TABLE 3 Form HA₀ XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 7.59 11.64 100 2 15.12 5.85 7.88 3 16.06 5.526.36 4 17.94 4.94 .5.41 5 23.89 3.72 7.95

Representative XRPD peaks for Form HC₀ are listed in the following Table4. The X-Ray diffraction pattern characteristic of Form HC₀ is shown inFIG. 4.

TABLE 4 Form HC₀ XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 7.49 11.79 10.37 2 8.36 10.56 100 3 8.7110.15 22.84 4 14.54 6.09 8.62 5 15.00 5.90 12.97 6 15.46 5.73 5.78 716.48 5.37 7.79 8 16.69 5.31 14.92 9 17.39 5.10 31.23 10 18.73 4.73 9.0011 19.79 4.48 8.55 12 20.69 4.29 7.10 13 23.36 3.81 5.86 14 23.53 3.785.43 15 24.59 3.62 43.43 16 25.42 3.50 13.96 17 26.04 3.42 5.27

Representative XRPD peaks for Form HD₀ are listed in the following Table5. The X-Ray diffraction pattern characteristic of Form HD₀ is shown inFIG. 5.

TABLE 5 Form HD₀ XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 7.60 11.62 100 2 8.99 9.83 5.05 3 15.16 5.8411.66

Representative XRPD peaks for the S2₀ form are listed in the followingTable 6. The X-Ray diffraction pattern characteristic of Form S2₀ isshown in FIG. 6.

TABLE 6 S2₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 8.56 10.32 58.9 2 9.80 9.02 58.8 3 10.62 8.3226.8 4 11.04 8.01 54.2 5 12.68 6.98 31.8 6 14.64 6.04 61.0 7 16.07 5.5181.0 8 17.18 5.16 37.5 9 17.23 5.14 43.7 10 19.75 4.49 50.9 11 22.243.99 100.0 12 23.02 3.86 99.5 13 23.31 3.81 22.8 14 27.06 3.29 55.8 1527.85 3.20 42.6

Representative XRPD peaks for the S3₀ form are listed in the followingTable 7. The X-Ray diffraction pattern characteristic of Form S3₀ isshown in FIG. 7.

TABLE 7 S3₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 6.70 13.19 54.7 2 7.61 11.61 12.5 3 8.6710.19 100.0 4 10.29 8.59 13.3 5 11.57 7.64 16.7 6 13.36 6.62 23.3 715.02 5.89 11.7 8 16.80 5.27 30.8 9 16.85 5.26 22.1 10 17.33 5.11 7.1 1125.20 3.53 7.1

Representative XRPD peaks for the S4₀ form are listed in the followingTable 8. The X-Ray diffraction pattern characteristic of Form S4₀ isshown in FIG. 8.

TABLE 8 S4₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 7.95 11.12 9.3 2 8.42 10.49 100.0 3 8.6010.28 21.9 4 13.92 6.36 17.4 5 17.20 5.15 14.1 6 21.07 4.21 5.9 7 21.304.17 6.4 8 24.46 3.64 17.3

Representative XRPD peaks for the S5₀ form are listed in the followingTable 9. The X-Ray diffraction pattern characteristic of Form S5₀ isshown in FIG. 9.

TABLE 9 S5₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 4.46 19.80 28.9 2 7.67 11.51 100.0 3 8.869.97 31.3 4 11.71 7.55 14.5

Representative XRPD peaks for the S6₀ form are listed in the followingTable 10. The X-Ray diffraction pattern characteristic of Form S6₀ isshown in FIG. 10.

TABLE 10 S6₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 8.36 10.57 19.7 2 8.68 10.18 100.0 3 11.107.97 46.3 4 15.42 5.74 15.7 5 16.21 5.46 21.6 6 16.94 5.23 33.2 7 17.255.14 14.7 8 17.39 5.10 29.0 9 23.31 3.81 71.4 10 26.27 3.39 23.7

Representative XRPD peaks for the S7₀ form are listed in the followingTable 11. The X-Ray diffraction pattern characteristic of Form S7₀ isshown in FIG. 11.

TABLE 11 S7₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 4.50 19.62 35.4 2 7.70 11.47 100.0 3 8.909.93 42.3 4 11.76 7.52 15.6

Representative XRPD peaks for the S9₀ form are listed in the followingTable 12. The X-Ray diffraction pattern characteristic of Form S9₀ isshown in FIG. 12.

TABLE 12 S9₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 8.34 10.59 100.0 2 8.67 10.19 23.0 3 16.685.31 7.2 4 17.33 5.11 8.5 5 24.57 3.62 39.3

Representative XRPD peaks for the S10₀ form are listed in the followingTable 13. The X-Ray diffraction pattern characteristic of Form S10₀ isshown in FIG. 13.

TABLE 13 S10₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 4.45 19.83 42.6 2 7.62 11.59 100.0 3 8.7910.06 43.4 4 11.62 7.61 14.7 5 15.84 5.59 11.3 6 17.67 5.02 12.9

Representative XRPD peaks for the S12₀ form are listed in the followingTable 14. The X-Ray diffraction pattern characteristic of Form S12₀ isshown in FIG. 14.

TABLE 14 S12₀ form XRPD peaks Peak No. Angle [°2 Theta] d-spacing[Angstrom] Intensity [%] 1 7.63 11.58 100 2 7.67 11.51 92 3 8.63 10.2412 4 9.00 9.82 55 5 14.78 5.99 10 6 17.13 5.17 12 7 17.39 5.09 11 817.99 4.93 33 9 18.15 4.88 10 10 24.46 3.64 60

Accordingly, in one aspect, the present invention pertains to acrystalline form of Compound I that is Form A₀, Form B₀, or a mixturethereof. In a further aspect, the crystalline form is Form A₀. Inanother aspect, the crystalline form is Form B₀. In a further aspect,the crystalline form is characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 4.32, 6.07, 8.55,12.07 and/or 15.37±0.2 degrees 2-theta. In yet another aspect, thecrystalline form has an X-ray powder diffraction pattern substantiallyas depicted in FIG. 1. In an additional aspect, the crystalline form ischaracterized by an X-ray powder diffraction pattern comprising one ormore of the following peaks: 7.16, 7.89, 10.77, 16.54, and/or 21.20±0.2degrees 2-theta. In still another aspect, the crystalline form has anX-ray powder diffraction pattern substantially as depicted in FIG. 2.

A further aspect of the present invention pertains to a crystalline formof Compound I that is Form HA₀, Form HC₀, Form HD₀ or a mixture thereof.In another aspect, the crystalline form is Form HA₀. In a furtheraspect, the crystalline form is Form HC₀. In an additional aspect, thecrystalline form is Form HD₀. In still another aspect, the crystallineform is characterized by an X-ray powder diffraction pattern comprisingone or more of the following peaks: 7.59, 15.12, 16.06, 17.94 and/or23.89±0.2 degrees 2-theta. In a further aspect, the crystalline form hasan X-ray powder diffraction pattern substantially as depicted in FIG. 3.In an additional aspect, the crystalline form is characterized by anX-ray powder diffraction pattern comprising one or more of the followingpeaks: 8.36, 8.71, 16.69, 17.39 and/or 24.59±0.2 degrees 2-theta. In yetanother aspect, the crystalline form has an X-ray powder diffractionpattern substantially as depicted in FIG. 4. In another aspect, thecrystalline form is characterized by an X-ray powder diffraction patterncomprising one or more of the following peaks: 7.60, 8.99 and/or15.16±0.2 degrees 2-theta. In a further aspect, the crystalline form hasan X-ray powder diffraction pattern substantially as depicted in FIG. 5.

Still another aspect of the present invention pertains to a crystallineform of Compound I that is Form S2₀, Form S3₀, Form S4₀, Form S5₀, FormS6₀, Form S7₀, Form S9₀, Form S10₀, Form S12₀ or a mixture thereof. In afurther aspect, the crystalline form is Form S2₀. In still anotheraspect, the crystalline form is Form S3₀. In an additional aspect, thecrystalline form is Form S4₀. In yet a further aspect, the crystallineform is Form S5₀. In still an additional aspect, the crystalline form isForm S6₀. In another aspect, the crystalline form is Form S7₀. In afurther aspect, the crystalline form is Form S9₀. In still anotheraspect, the crystalline form is Form S10₀. In a further aspect, thecrystalline form is Form S12₀. In a further aspect, the crystalline formis characterized by an X-ray powder diffraction pattern comprising oneor more of the following peaks: 8.56, 14.64, 16.07, 22.24 and/or23.02±0.2 degrees 2-theta. In yet another aspect, the crystalline formis characterized by an X-ray powder diffraction pattern comprising oneor more of the following peaks: 6.70, 8.67, 13.36, 16.80 and/or16.85±0.2 degrees 2-theta. In an additional aspect, the crystalline formis characterized by an X-ray powder diffraction pattern comprising oneor more of the following peaks: 8.42, 8.60, 13.92, 17.20 and/or24.46±0.2 degrees 2-theta. In another aspect, the crystalline form ischaracterized by an X-ray powder diffraction pattern comprising one ormore of the following peaks: 4.46, 7.67, 8.86 and/or 11.71±0.2 degrees2-theta. In an additional aspect, the crystalline form is characterizedby an X-ray powder diffraction pattern comprising one or more of thefollowing peaks: 8.68, 11.10, 16.94, 17.39 and/or 23.31±0.2 degrees2-theta. In another aspect, the crystalline form is characterized by anX-ray powder diffraction pattern comprising one or more of the followingpeaks: 4.50, 7.70, 8.90 and/or 11.76±0.2 degrees 2-theta. In stillanother aspect, the crystalline form is characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.34,8.67, 16.68, 17.33 and/or 24.57±0.2 degrees 2-theta. In an additionalaspect, the crystalline form is characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 4.45,7.62, 8.79, 11.62 and/or 17.67±0.2 degrees 2-theta. In another aspect,the crystalline form is characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 7.63, 7.67, 9.00,17.99 and 24.46±0.2 degrees 2-theta.

An additional aspect of the present invention pertains to a process forpreparing a crystalline form of Compound I that is Form A₀, comprisingthe steps of: (a) slurrying Compound I in hydrocarbons (such as heptaneor toluene); (b) cooling the resulting slurry; (c) filtering theresulting slurry; and (d) drying the filter-cake. In one aspect,Compound I is slurried in 26 to 45 volumes of heptane. In anotheraspect, Compound I is slurried in 45 volumes of heptane. In anadditional aspect, step (a) is performed at 79 to 83° C. In stillanother aspect, step (a) is performed at 85° C. In yet another aspect,step (a) is performed for 24 to 48 hours. In a further aspect, step (a)is performed for 45 hours. In another aspect, step (b) occurs at atemperature of 30-65° C.

In still another aspect, step (b) is performed at 65° C. In anadditional aspect, step (d) is performed at room temperature for 0.33 to3 hours. In still another aspect, step (d) is performed at roomtemperature for three hours.

A further aspect of the present invention pertains to a process forpreparing a crystalline form of Compound I that is Form A₀, comprisingthe steps of: (a) dissolving Compound I in a solvent; (b) filtering theresulting solution; (c) partially distilling the solvent while adding ananti-solvent to precipitate Compound I; (d) further distilling theresulting slurry while adding additional anti-solvent to reduce thevolume of the solvent used in step (a); (e) heating the slurry toachieve complete conversion to Form A₀; (f) cooling; (g) collecting theproduct via filtration; and (h) drying. In a further aspect, step (a) isperformed using 27 to 35 volumes of THF. In another aspect, step (a) isperformed using 30 volumes of THF. In a further aspect, the solutionproduced via step (a) may optionally be treated with a metal scavengeror carbon. In still a further aspect, the filtering step (b) comprisesone or both of the following steps: (i) filtering to remove the metalscavenger; and (ii) polish filtering through a 1-micron inline cartridgefilter. In a further aspect, the solvent present in step (c) isdistilled to 60 to 90% of its original volume. In an additional aspect,step (c) is performed using a hydrocarbon (such as heptane) as theanti-solvent. In another aspect, step (d) is performed until less than5% THF by volume remains. In still another aspect, step (e) is performedat a temperature of about 90 to 96° C. In an additional aspect, step (e)may be optionally omitted. In another aspect, the slurry is agitated forabout 3 to 5 hours. In a further aspect, step (f) is performed atambient temperature (25±5° C.). In an additional aspect, the filtrationof step (g) is performed using a dry, inert gas. In another aspect, step(h) is performed at a temperature up to 80° C. In yet another aspect,the residual water and/or solvate(s) are azeotropically removed.

Yet another aspect of the present invention pertains to a pharmaceuticalcomposition comprising Form A₀, Form B₀, Form HA₀, Form HC₀ or Form HD₀,or a mixture thereof A further aspect pertains to a method of treatingcancer comprising the step of administering to a patient in need thereofa therapeutically effective amount of a pharmaceutical compositioncomprising Form A₀, Form B₀, Form HA₀, Form HC₀ or Form HD₀ or a mixturethereof. In an additional aspect, the present invention pertains to amethod of treating cancer comprising the step of administering to apatient in need thereof a therapeutically effective amount of apharmaceutical composition comprising Form A₀.

TERMINOLOGY

The term “amorphous,” as used herein, means lacking a characteristiccrystal shape or crystalline structure.

The term “anti-solvent,” as used herein, means a solvent in which acompound is substantially insoluble.

The term “crystalline,” as used herein, means having a regularlyrepeating arrangement of molecules or external face planes.

The term “crystalline form,” as used in herein, refers to a solidchemical compound or mixture of compounds that provides a characteristicpattern of peaks when analyzed by x-ray powder diffraction; thisincludes, but is not limited to, polymorphs, solvates, hydrates,co-crystals, and de-solvated solvates.

The term “polymorphic” or “polymorphism” is defined as the possibilityof at least two different crystalline arrangements for the same chemicalmolecule.

The term “solute” as used herein, refers to a substance dissolved inanother substance, usually the component of a solution present in thelesser amount.

The term “solution,” as used herein, refers to a mixture containing atleast one solvent and at least one compound at least partially dissolvedin the solvent.

The term “solvate,” as used herein, refers to a crystalline materialthat contains solvent molecules within the crystal structure.

The term “solvent,” as used herein, means a substance, typically aliquid, that is capable of completely or partially dissolving anothersubstance, typically a solid. Solvents for the practice of thisinvention include, but are not limited to, water, acetic acid, acetone,acetonitrile (ACN), benzyl alcohol, 1-butanol, 2-butanol, 2-butanone,butyronitrile, tert-butanol, N-butyl acetate, chlorobenzene, chloroform,cyclohexane, 1-2 dichloloroethane (DCE), dichloromethane (DCM),diethylene glycol dibutyl ether (DGDE), diisopropyl amine (DIPA),diisopropyl ether (DIPE), 1,2-dimethoxyethane (DE),N,N-dimethylacetamide (DMA), 4-dimethylaminopyridine (DMAP),N,N-dimethylformamide (DMF), dimethyl sulfoxide, 1,4-dioxane,ethyleneglycoldiemethylether, ethanol, ethyl acetate,ethyldiisopropylamine, ethylene glycol, ethyl formate, formic acid,heptane, isobutyl alcohol, isopropyl acetate (IPAC), isopropyl alcohol(IPA), isopropyl amine, lithium diisopropylamide (LDA), methanol,methoxy benzene (MTB), methyl acetate, methyl ethyl ketone (MEK), methylisobutyl ketone (MIK), 2-methyltetrahydrofuran, methyl tert-butyl ether(MTBE), 1:1 formamide:water, 1:1 N-methylpyrrolidinone (NMP): water,2-pentanone, 3-pentanone, 1-pentanol, 1,2-propanediol, 2-propanol (IPA),1-propanol, propanonitrile, propylene carbonate, 1,2-propylene glycol(PG), pyridine, tetrahydrofuran (THF), tetrahydropyran (THP), toluene,triethyl amine, xylene, mixtures thereof and the like. These solventsare categorized into five classes according to their functional group:Class 1: “Protic” or hydrogen bond donating solvents (Lewis acids),including benzyl alcohol, ethanol, IPA, methanol, and water; Class 2:Hydrogen bonding acceptor solvents (Lewis bases), including acetone,1,4-dioxane, DMF, ethyl acetate, MEK, MTBE, THF, and water; Class 3:Polar aprotic solvents, better termed “nonhydroxylic solvents,”including acetonitrile, DMA, DMF, and DMSO; Class 4: Chlorocarbonsolvents, which include chloroform; Class 5: Hydrocarbon solvents, bothsaturated and unsaturated, including n-heptane, toluene, p-xylene, andxylene.

The term “therapeutically effective amount,” as used herein, refers tothe amount determined to be required to produce the physiological effectintended and associated with a given drug, as measured according toestablished pharmacokinetic methods and techniques, for the givenadministration route. Appropriate and specific therapeutically effectiveamounts can be readily determined by the attending diagnostician, as oneskilled in the art, by the use of conventional techniques. The effectivedose will vary depending upon a number of factors, including the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, the formulation of the active agent withappropriate excipients, and the route of administration. Typically, thecrystalline forms would be administered at lower dosage levels, with agradual increase until the desired effect is achieved.

Unless stated otherwise, percentages stated throughout thisspecification are weight/weight (w/w) percentages.

The term “pharmaceutically acceptable excipients,” as used herein,includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutical activesubstances is well known in the art, such as in Remington: The Scienceand Practice of Pharmacy, 20^(th) ed.; Gennaro, A. R., Ed.; LippincottWilliams & Wilkins: Philadelphia, Pa., 2000. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

For therapeutic purposes, the crystalline forms of the present inventioncan be administered by any means that results in the contact of theactive agent with the agent's site of action in the body of the subject.The crystalline forms may be administered by any conventional meansavailable for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in combination with other therapeuticagents, such as, for example, analgesics. The crystalline forms of thepresent invention are preferably administered in therapeuticallyeffective amounts for the treatment of the diseases and disordersdescribed herein to a subject in need thereof.

In therapeutic or prophylactic use, the crystalline forms of the presentinvention may be administered by any route that drugs are conventionallyadministered. Such routes of administration include intraperitoneal,intravenous, intramuscular, subcutaneous, intrathecal, intracheal,intraventricular, oral, buccal, rectal, parenteral, intranasal,transdermal or intradermal. Administration may be systemic or localized.

The crystalline forms described herein may be administered in pure form,combined with other active ingredients, or combined withpharmaceutically acceptable nontoxic excipients or carriers. Oralcompositions will generally include an inert diluent carrier or anedible carrier. Pharmaceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a dispersing agent such as alginic acid, Primogel,or corn starch; a lubricant such as magnesium stearate; a glidant suchas colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials that modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents. Further, a syrupmay contain, in addition to the active compounds, sucrose as asweetening agent and certain preservatives, dyes, colorings, andflavorings.

Alternative preparations for administration include sterile aqueous ornonaqueous solutions, suspensions, and emulsions. Examples of nonaqueoussolvents are dimethylsulfoxide, alcohols, propylene glycol, polyethyleneglycol, vegetable oils such as olive oil and injectable organic esterssuch as ethyl oleate. Aqueous carriers include mixtures of alcohols andwater, buffered media, and saline. Intravenous vehicles include fluidand nutrient replenishers, electrolyte replenishers, such as those basedon Ringer's dextrose, and the like. Preservatives and other additivesmay also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, inert gases, and the like.

Preferred methods of administration of the crystalline forms to mammalsinclude intraperitoneal injection, intramuscular injection, andintravenous infusion. Various liquid formulations are possible for thesedelivery methods, including saline, alcohol, DMSO, and water basedsolutions. The concentration may vary according to dose and volume to bedelivered and can range from about 1 to about 1000 mg/mL. Otherconstituents of the liquid formulations can include preservatives,inorganic salts, acids, bases, buffers, nutrients, vitamins, or otherpharmaceuticals such as analgesics or additional PARP and kinaseinhibitors.

Instrumentation

X-Ray Powder Diffraction (XRPD)

XRPD patterns were recorded on a PANalytical X′ Pert Pro diffractometerusing Cu Kα radiation at 40 kV and 40 mA. A silicon standard was run tocheck the x-ray tube alignment. The sample was pressed onto azero-background quartz plate in an aluminum holder. The standard X-raypowder pattern scans were collected from ca. 2 to 40° 20 with a 0.0080°step size and 96.06 sec counting time which resulted in a scan rate ofapproximately 0.5°/min.

For the single crystal studies, the crystals chosen were coated withparatone oil and flash frozen on an Oxford diffraction CCDdiffractometer (Xcalibur S, with a Sapphire detector). Data werecollected with standard area detector techniques. The structures weresolved and refined with the SHELXTL package. Default Reitveld refinementof the single crystal parameters against the measured XRPD pattern gavea good fit with no unexplained peaks.

Variable Temperature X-Ray Powder Diffraction (VT-XRPD)

Variable temperature studies were performed under a nitrogen atmospherewith an Anton Paar TTK450 temperature chamber under computer controlthrough an Anton Paar TCU100 temperature control unit. Two measurementschemes were used, restricted and continuous. In the restricted mode,measurements were made, only after the TK450 chamber reached therequested temperature. In the continuous mode, the sample was heated at10° C./minute and fast scans were measured as the temperature changed.After the requested temperature was reached, the sample was cooled at 30or 35° C./minute and a slower scan measured 25° C. The temperatureschosen were based on DSC results.

Differential Scanning Calorimetry (DSC)

Thermal curves were acquired using a Perkin-Elmer Sapphire DSC unitequipped with an autosampler running Pyris software version 6.0calibrated with indium prior to analysis. Solid samples of 1-11 mg wereweighed into 20 μL aluminum open sample pans. The DSC cell was thenpurged with nitrogen and the temperature heated from 0° to 275° C. at10° C./min.

Thermogravimetric Analysis (TGA)

Thermal curves were acquired using a Perkin-Elmer Pyris 1 TGA unitrunning Pyris software version 6.0 calibrated with calcium oxalatemonohydrate. TGA samples between 1-15 mg were monitored for percentweight loss as heated from 25° to 400° C. at 10° C./min in a furnacepurged with helium at ca. 50 mL/min.

Dynamic Vapor Sorption (DVS)

Gravimetric vapor sorption experiments were carried out using the DVS-HTinstrument (Surface Measurement Systems, London, UK). This instrumentmeasures the uptake and loss of vapor gravimetrically using a recordingultra-microbalance with a mass resolution of ±0.1 μg. The vapor partialpressure (±1.0%) around the sample was controlled by mixing saturatedand dry carrier gas streams using electronic mass flow controllers. Thedesired temperature was maintained at ±0.1° C.

The samples (10-25 mg) were placed into the DVS-HT instrument at thedesired temperature. Two types of dynamic vapor sorption experimentswere performed:

-   -   1. The sample was initially dried in stream of dry air (<0.1%        relative humidity (RH)) for 20 hours to establish a dry mass and        exposed to two 0-90% RH cycles (in 10% RH increments).    -   2. The sample was exposed at 90% RH for 20 hours and exposed to        two 90-0% RH cycles (in 10% RH increments).        Infrared Spectrometry (FTIR)

Spectra were obtained using a Thermo Electron-Nicolet Avatar 370 DTGSinstrument with the Smart Orbit ATR attachment containing a diamondcrystal window. Thermo Electron Omnic™ software (version 3.1) was usedto compute the spectrum from 4000 to 400 cm⁻¹ from the initialinterferogram. A background scan was collected before obtaining eachsample spectrum. For each sample, 32 scans were obtained at 4 cm⁻¹spectral resolution and averaged.

Raman Spectrometry

The Raman spectra of the sample were recorded with a FT-Raman module ona vertex 70 FTIR spectrometer (Bruker RAM II, Bruker optics, Germany). Agermanium photodiode was used to record FT-Raman spectra excited by anNd:Yag laser (suppression of fluorescence). A polystyrene standard wasrun prior to sample analyses. Acquisition time for each spectrum was 1minute, with a resolution of 4 cm⁻¹ and the power of the 1064 nm laserat the sample was 50 mW.

Identity, Assay and Purity

Typically 10 μL aliquots of the sample solutions were diluted to 1 mLwith acetonitrile and the assay concentrations were determined from anaverage of duplicate injections using the following HPLC method. Thepurity and impurity analyses are done using conventional HPLC.

Column: Zorbax Eclipse XDB C₁₈, 4.6×150 mm, 5μ

Column temperature: 25° C.

Injection volume: 5 μL

Detection: UV, 238 nm

Flow rate: 0.8 mL/min

Run time: 30 minutes

Mobile phase A: 0.1% TFA in water

Mobile phase B: 0.1% TFA in acetonitrile

Time (min) % A % B 0 70 30 6.0 55 45 10 55 45 25 10 90 25.1 70 30 30 7030

EXAMPLES Process for Preparing Compound I

Compound I can be prepared according to Scheme I:

In Scheme I, the synthesis is initiated with 4-methoxyindole, acommercially available starting material. Upon masking the indolenitrogen with di-tert-butyldicarbonate ((Boc)₂O), the indole derivativeis activated with lithium diisopropylamide (LDA) to generate thecarbanion at the 2-position of the indole, which reacts in-situ withtriisopropyl borate. Acidic workup hydrolyzes the boronate esterintermediate to the corresponding indole boronic acid compound A.Compound A is then coupled with 1-cyclopentenyltrifluoromethanesulfonate (also called enol triflate in this report) inthe presence of catalytic amounts of palladium acetate andtriphenylphoshpine under Suzuki conditions to give the key dieneintermediate compound B. After removing the Boc protecting group withsodium methoxide, diene compound C is coupled with maleimide viaDiels-Alder reaction in acetic acid to give the pentacyclic intermediatecompound D. Aromatization via chloranil oxidation converts compound D tocompound E, which is coupled with 1-methylpiperazine under Mannichconditions to furnish the target molecule Compound I. Detailed aspectsof the synthesis are provided below.

Synthesis of N-Boc-4-Methoxyindole

Into a 100-gal glass-lined reactor was charged 4-methoxyindole (20.0 kg,136 mol, Yixing Zhongyu Medicine Technology Co., Ltd.), followed by DMAP(0.50 kg, 4.1 mol, Aldrich) and toluene (92 kg, Corco reagent grade).The resulting mixture was stirred and warmed to about 40° C. Meanwhile,a solution of di-tert-butyl dicarbonate (31.8 kg, 146 mol, LacamasLaboratories, Inc.) in toluene (60 kg, Corco reagent grade) was preparedin a second reactor. This solution was added to the indole solution overabout 1³/4 hours. The slightly exothermic reaction (maximum temperatureabout 41° C.) was accompanied by gas evolution. After being agitated foran additional hour at 40° C. the reaction solution was cooled to 20±3°C. An in-process test revealed that 4-methoxyindole was consumedcompletely. Deionized water (15 gallons) was added to decompose theexcess (Boc)₂O (Caution: gas evolution). The resulting mixture wasagitated vigorously for ½ hour then allowed to stand overnight. Afterthe lower aqueous layer was removed, the organic layer was partiallyconcentrated under reduced pressure to remove about 145 L of distillate(60° C. jacket, up to 60 mmHg). At this point, additional toluene (30kg, Corco reagent grade) was charged in and distillation continued untila total of approximately 200 L of distillate was collected. The batchwas then cooled to room temperature and drained into a poly drum,resulting in 62.3 kg of a dark amber solution containing 33.6 kg ofN-Boc-4-methoxyindole (theoretical yield assumed). This was used in thenext stage without further purification.

Synthesis of Compound A 2-Borono-4-methoxy-1H-indole-1-carboxylic acid1-(1,1-dimethylethyl) ester)

Approximately half of the above solution was charged into a 100-galglass-lined reactor, followed by the additions of toluene (3.0 kg todilute the charge to 50 wt %), triisopropyl borate (19.9 kg, 105.9moles, Anderson Development Co.), and THF (91 kg, Corco reagent grade).The resulting solution was agitated and cooled to −2° C. At this point,lithium diisopropylamide (37.3 kg, 91.8 moles, 27% solution in ethylbenzene/tetrahydrofuran/heptane, FMC Lithium) was added over one hour,keeping the batch temperature below 3° C. (−10° C. jacket). Theresulting reaction mixture was agitated at 0±3° C. after the addition ofLDA until the completion of the reaction was detected by HPLC (0.6 A %of N-Boc-4-methoxyindole remaining 30 min after the addition of LDA).Meanwhile, a solution of 3 N HCl was prepared and cooled to ˜5° C. in asecond reactor by diluting 27 kg of concentrated hydrochloric acid in16.3 gallons of de-ionized water. This dilute HCl was added to the batchover one hour to maintain the batch temperature at <15° C. (Batchtemperature reached 8° C. at the end of the addition.) The jackettemperature was then set to 20° C. The reactor and addition lines wererinsed with deionized water (6 gallons) and the rinse was combined withthe batch. This was followed by the addition of MTBE (27 kg, Pride). Theresulting mixture was agitated for ½ hour then stopped for phaseseparation. The aqueous layer was separated and back-extracted with MTBE(14 kg, Pride) in a second reactor. The combined organic layers werewashed consecutively with 5% NaCl (34 L), 5% NaHCO₃ (34 L), and 10% NaCl(19 L). After being dropped to a drum and weighed (172.2 kg), theorganic phase was returned to the reactor and concentrated under reducedpressure (reactor jacket set point: 30° C.), removing 116 kg ofdistillate over a three-hour period. The resulting slurry was dilutedwith n-heptane (75 kg, CORCO reagent grade) and further distilled toremove additional 75 L of distillate. After being stirred at roomtemperature overnight, the slurry was cooled to ˜5° C. for one hour. Theproduct was collected on an Aurora filter and washed with 33 kg ofn-heptane. The filter cake was tray-dried under house vacuum overnightwith nitrogen bleeding but no heat. There resulted 17.8 kg (88.8% yield,corrected) of compound A as an off-white solid. HPLC purity: 100 LCAP,95.8 LCWP.

Synthesis of 1,1,1-trifluoromethanesulfonic acid 1-cyclopenten-1-ylester

Into a 100-gallon glass-lined reactor at room temperature was chargedcyclopentanone (8.95 kg, 106.5 mol), followed by toluene (116.40 kg,CORCO reagent grade) and ethyldiisopropylamine (16.6 kg, 128.7 mol). Theresulting solution was agitated and heated to 45±5° C. At this point,trifluoromethanesulfonic anhydride (36.2 kg, 128.4 mol) was added overapproximately one hour from a 30-L addition flask. The addition oftrifluoromethanesulfonic anhydride was very exothermic. Jacket cooling(set at 10° C.) was applied to maintain the batch temperature at 45±5°C. The batch did fall below 40° C. for 7 minutes during the 44 minuteaddition. Agitation continued at 39-45° C. for 20 minutes after theaddition of trifluoromethanesulfonic anhydride. An in-process test afterthis 20 minutes revealed the total consumption of cyclopentanone. Afterbeing cooled to 19.6° C., the batch was filtered through a pad of Celite(18.0 kg) in a filter. The filtrate was collected in a clean poly-linedsteel drum. The Celite pad was rinsed with toluene (37.0 kg, CORCOreagent grade). The rinse was combined with the batch in the samepoly-lined steel drum. The filtrate (159.85 kg) was analyzed against areference standard to show it contained 19.50 kg (83.3% yield) of enoltriflate. This enol triflate solution in toluene was kept in the coldroom overnight and used in the subsequent Suzuki coupling withoutfurther purification.

Synthesis of Compound B2-(1-Cyclopenten-1-yl)-4-methoxy-1H-indole-1-carboxylic acid1,1-dimethylethylester

Into a 100-gal glass-lined reactor at room temperature were chargedcompound A (18.00 kg, 61.8 mol), triphenylphosphine (648.8 g, 2.47 mol),and palladium acetate (277.0 g, 1.23 mol). The reactor was thenevacuated and refilled with nitrogen three times. Toluene (78.3 kg,CORCO reagent grade) was jet pumped into the reactor followed bydicyclohexylamine (44.5 kg, 245.4 mol). This addition took 4 minutes.The resulting slurry was allowed to agitated vigorously (125 rpm) atroom temperature for 21 minutes, followed by the slow addition of enoltriflate stream in toluene (131.7 kg, containing 16.07 kg of enoltriflate, 74.3 mol) over 43 minutes. The addition of enol triflate wasexothermic. Jacket cooling was applied to keep the batch temperature at18.8-27.5° C. The resulting heterogeneous mixture was agitated at18.4-22.3° C. until the completion of the reaction was detected by HPLC(Note: Though the reaction was complete in less than an hour it wasstill agitated at the room temperature overnight before continuing thework up. This was strictly for the sake of convenience. The batch may beheld at room temperature for up to 100 hours with no adverse effect onthe product.) Celite (9.00 kg) was added to the batch. The batch wasagitated at room temperature for 10 minutes, and then filtered through apad of Celite (2.70 kg) in a filter. The filtrate was collected in twoclean poly-lined steel drums. The filter cake was rinsed with toluene(47.8 kg, CORCO reagent grade). The rinse was combined with the batch inthe same poly-lined steel drums. The filtrate (260.45 kg) was analyzedagainst a reference standard to show it contained 20.59 kg (106.4%yield) of compound B. It was assumed based on the assay that thisreaction went in 100% yield, and the charges for the next step were doneas if it had gone in 100% yield. The solution of compound B in toluenewas kept in the pilot plant at room temperature and used in thesubsequent deprotection procedures without further purification.

Synthesis of Compound C 2-(Cyclopenten-1-yl)-4-methoxy-1H-indole

Into a 100-gal glass-lined reactor at room temperature was charged thetoluene stream of compound B (12.82 kg of compound B, 40.96 moles),followed by the addition of sodium methoxide (44.0 kg, 25-30 wt %solution in MeOH, 203.7 moles). The resulting solution was agitated andheated to 45±5° C. Agitation continued at 45±5° C. until the completionof the reaction was detected by HPLC (reaction complete in ˜4 hrs, HPLCdata returned at ˜8 hrs). The batch was then cooled to 23.5° C. over 26minutes. The batch was agitated overnight at 22±2° C. After ˜17 hours at22° C. approximately ½ of the batch (111.15 kg) was transferred to asecond reactor and worked up separately. To the first reactor wascharged DI water (21 gallons). The resulting mixture was agitated for 16minutes then stopped. After the batch was allowed to settle at roomtemperature for 46 minutes, the bottom aqueous layer was removed. Thiswas followed by a small portion of a rag layer that was drained into aclean carboy. The remaining organic layer was filtered through a pad ofCelite (3.84 kg) in a filter. The filtrate was collected in a cleanpoly-lined steel drum. The rag layer was then filtered through the samecelite pad and the filtrate was collected in a new carboy. The Celitepad was washed with toluene (6.20 kg, CORCO reagent grade) and this washwas combined with the filtered rag layer. The filtered rag layer wasthen transferred to a glass addition vessel where the bottom aqueouslayer was removed and the organic layer from the rag was combined withthe original organic layer. The above workup procedure was repeated onthe second half of the batch, generating the second toluene solution ofcompound C. As much of the second solution as would fit was placed inthe poly-lined steel drum with the first organic layer (164.70 kg,containing 8.14 kg of compound C). The remaining second organic layerwas contained in a small poly drum (19.05 kg, containing 0.49 kg ofcompound C). These two solutions were held in the pilot plant forfurther processing in the next stage without any further purification. Atotal of 8.63 kg (99.2% yield) of compound C was generated.

Synthesis of Compound D3a,3b,4,5,6,6a,7,11c-Octahydro-[1-methoxy-1H-cyclopentaia]pyrrolo[3,4-c]carbazole-1,3(2H)-dione

Into a 100-gal glass-lined reactor at room temperature was charged atoluene stream of compound C (12.58 kg of compound C, 59.1 mol). Thissolution was concentrated under full house vacuum and <40° C. internaltemperature until the residue was approximately six times the weight ofcompound C (targeted volume ˜75.5 L) over approximately 7 hours. Thisresidue was drained into a clean polyethylene drum and used in thefollowing Diels-Alder reaction without any further purification. Into asecond 100-gal glass-lined reactor was charged maleimide (7.45 kg, 76.8mol, Carbosynth Limited), followed by glacial acetic acid (145.70 kg).The resulting mixture was stirred to achieve a solution. At this point,the concentrated compound C solution from above (84.95 kg) was chargedin over approximately 20 minutes to control the batch temperature at20±10° C. (Jacket temperature was set at 15° C.) The resulting mixturewas agitated at 30±3° C. until the completion of the reaction wasdetected by HPLC (reaction is done at ˜15.5 hours, HPLC data is receivedat ˜17.5 hours). The batch was then cooled to 23.2° C. overapproximately 20 minutes. After the mother liquor was analyzed by aweight based HPLC assay and confirmed that it contained less than 10% ofcompound D (found: 5.88%), the batch was filtered on an Aurora filter(2.5 hrs from reaching 23.2° C. to filter time). The filter cake wasrinsed with glacial acetic acid (39.65 kg) and pulled dry in the filterunder vacuum with a stream of nitrogen until the purity of compound Dmet the set specification (>90 wt %) by HPLC weight based assay (dryingwas done over 3 nights, purity was 99.5 wt % after 3 nights). Theproduct was then unloaded to a double polyethylene bag-lined fiber drumto give 13.43 kg (73.3% yield) desired compound D as a tan solid. Thismaterial was used in the subsequent chloranil oxidation without anyfurther purification.

Synthesis of Compound E4,5,6,7-tetrahydro-11-methoxy-1H-cyclopenta[a]pyrrolo[3,4-c]carbazole-1,3(2H)dione

Into a 100-gal glass-lined reactor at room temperature was chargedcompound D (28.66 kg, 92.45 mol), followed by tetrachloro-p-benzoquinone(45.50 kg, 185.0 mol, 99%, ACROS) and THF (253.1 kg, CORCO reagentgrade). The resulting heterogeneous mixture was heated to 65±5° C. andagitated at this temperature until the completion of the reaction wasdetected by HPLC (reaction is done in ˜22 hrs, HPLC data is received at˜23 hrs). The batch was then cooled to 22±5° C. over 35 minutes,analyzed for the loss of compound E in the solution (<10% specification.found: 1.9%), and filtered on a filter. The reactor, lines, and filtercake were rinsed with a mixture of THF-EtOH—H₂O (prepared in a secondreactor by mixing 62.0 kg of THF with 41.25 kg of EtOH and 4.64 gallonsof de-ionized water). The wet cake was dried in a filter under vacuumwith a stream of nitrogen until the product meets the set specification(>80 wt % of compound E spec. found: 80.8 wt % after 5 days). Theproduct was then unloaded to two double polyethylene bag-lined plasticpails, yielding 23.84 kg (86.7% yield) of compound E as a dark greenishyellow solid. This material was used directly in the subsequent Mannichreaction without further purification.

Synthesis of Compound I

Into a 100-gal glass-lined reactor was charged compound E (15.20 kg,40.1 moles), followed by paraformaldehyde (2.51 kg, 80.9 moles, 97%,ACROS) and denatured ethanol (223.45 kg, reagent grade). The resultingmixture was agitated (121 rpm) while 1-methylpiperazine (6.65 kg, 65.77moles, ACROS, 99%) was added over approximately 10 min from an additionflask. The resulting reaction mixture was heated and agitated at 70° C.The progress of the reaction was monitored by HPLC (1.35 A % compound Eremaining after ˜5 hours). After being agitated at 70° C. for a total of9 hours, the batch was cooled to 20±3° C. and stirred at thistemperature overnight. The product was filtered on a filter. The filtercake was rinsed with ethanol (43.9 kg, reagent grade) and pulled dry onthe filter with nitrogen bleeding until the residual ethanol was lessthan 12 wt % by ¹H NMR (8.4 wt % vs. compound I). The product was thenunloaded to a polyethylene bag-lined fiber drum to give 18.05 kg (95.8%yield) of crude compound I as a yellow solid: 98.6 LCAP, 89.2 LCWP. Thismaterial was used directly in the down stream process without furtherpurification.

Polymorph Screening Studies

Crystallization studies were performed to investigate polymorphism in 48different solvents. Solvents were selected on the basis of acceptability(ICH Class 3 and 2) and to give a range of dielectric constants, dipolemoments and functional groups. Two starting materials were selected:Form A₀ and Lot 7 (a mixture of Form A₀, Form HC₀ and Form HD₀). Whenpossible, full characterization was performed on the new forms that weregenerated during the polymorphism screening of Compound I. Thischaracterization consisted of: XRPD, thermal analysis, DVS, storage at40° C./75% RH and purity.

Four crystallization procedures including cooling, evaporation andanti-solvent addition were employed to obtain different polymorphicforms of Compound I. The details of each crystallization procedure aregiven below. The solid forms obtained from each solvent from theseprocedures are summarized in Table 15.

Crystallization Procedure:

1. Rapid Crystallization Screen

Two small scale screening procedures were used:

A. Approximately 1 mg of Compound I was weighed into a 0.5 mLpolypropylene centrifuge tube and 0.5 mL of a solvent. The centrifugetube was allowed to stand for 18 hours undisturbed at room temperatureand observed for changes. The tube was then agitated for 2.5 hours at52.5° C. and each tube observed for changes. The warmed centrifuge tubewas then agitated for 20 hours at 2-8° C. and observations made forchanges in crystallinity (if any) from initial room temperaturecondition were recorded.

B. Plates containing 10 volumes of Compound I (40 mg of Lot 7 in 400 μL)were heated from 20° C. to an initial temperature of 80° C. at a rate of4.8° C./min and after 30 minutes, cooled at a slow (0.28° C./min), orfast (10° C./min) rate to a final temperature of 5° C. and kept at thattemperature for 18 hours. The crystallization experiments were carriedout in glass vial (4 mL) well plates, and solid material was isolated byfiltration. The solid was dried at 57° C. for 10 hours.

2. Quick Cool Crystallization

Samples were prepared by adding 40 mg (±2) of Compound I solid materialinto a solvent volume to assure saturated conditions at the boilingpoint. The mixture was cooled and filtered through a 0.2μ nylon membranefilter into a warmed glass vial or Erlenmeyer flask. The solution wascooled to room temperature and placed in a refrigerator (ca. 4° C.)until crystal formation appeared to reach completion as determined byvisual inspection. Each refrigerator-sample was decanted and thecrystals were transferred to weighing paper and dried to constant weightunder ambient laboratory conditions. Samples difficult to decant werecentrifuged at 12000 rpm for four minutes. If the quick-cool proceduredid not result in solid materials, these samples were concentrated byevaporating approximately half the solvent volume. The solutions wereagain placed in the refrigerator and any solid material was isolated bydecanting or centrifugation.

3. Crystallization by Maturation with Lot 7 and Form A₀

Two types of maturation studies were performed:

A. Samples were prepared by adding approximately 10 mg of either Lot 7or Form A₀ to 1.0 mL of each solvent in a screw cap vial (about 4.0 mLvolume). These were then warmed to 64° C. while being shaken. Afterholding at 64° C. for 40 minutes, the samples were chilled down to 5° C.(at a rate of −0.25° C./min). The samples were held at 5° C. for a totalof 18 hours and transferred via pipette to 1.5 mL polypropylenecentrifuge tubes and spun at 12000 RPM for 1 minute. The supernatantliquid was decanted. The residues in the centrifuge tubes or glass vialswere then dried in a vacuum drying oven at 110° C. for 18 hours andanalyzed by XRPD.

B. Approximately 40 mg of Form A₀ was slurried in the different solvents(10 volumes (40 mg in 400 μL). The slurries were shaken for 48 hourswith alternating 4 hour periods at 50° C. (0.5° C./min) and 5° C. (−0.5°C./min). Any solid material was then isolated by filtration and analyzedby XRPD and thermal analysis.

4. Crystallization by Slurry with Form A₀

The slurries (20 mg of form A₀ in 500 μL of each solvent) were shaken at25° C. with different times. The solid was isolated by filtration anddried at 57° C. for 2 hours and analyzed by XRPD.

The XRPD results from the isolated solids from the four crystallizationmethods are recorded in Table 15 below.

TABLE 15 Summary of forms of Compound I obtained based on XRPD resultsfrom 48 different solvents and different crystallization methods SolventForms Obtained by XRPD 1,2-dichloroethane A₀, HA₀, HC₀, HD₀1,2-dimethoxyethane HA₀ 1,4-dioxane A₀, HC₀, HD₀ 1-butanol HD₀1-pentanol HD₀ 1-propanol S9₀, HC₀, HD₀ 2-butanol A₀, HC₀, HD₀2-butanone HC₀, HD₀ 2-methyl-tetrahydrofuran A₀, HC₀, HD₀ 2-pentanoneHA₀, HC₀, HD₀ 2-propanol S3₀, HC₀, HD₀ 3-pentanone A₀, HA₀, HC₀, HD₀Acetone A₀, HC₀, HD₀ Acetonitrile HC₀, HD₀ Butyronitrile A₀, HA₀, HDChlorobenzene A₀, HD₀ Chloroform HC, HD₀ Cyclohexane A₀, HC₀, HD₀Dichloromethane A₀, HC₀, HD₀ Diethylene glycol dibutyl ether A₀Diisopropyl amine A₀, HD₀ Diisopropyl ether A₀, HA₀, HC₀, HD₀ Dimethylsulfoxide HC₀, HD₀ Ethanol S4₀ Ethyl acetate A₀, HC₀, HD₀ Ethyl formateHA₀ Ethylene glycol S6₀ Heptane A₀, HC₀, HD₀ Isobutanol S12₀, HD₀Isopropyl acetate A₀, HC₀, HD₀ Methanol S2₀ Methoxybenzene HD₀ Methylacetate A₀, HA₀, HC₀, HD₀ Methyl isobutyl ketone A₀, HC₀, HD₀ Methyltert-butyl ether A₀, HA₀, HC₀, HD₀ N,N-dimethylacetamide S10₀N,N-dimethylformamide S5₀ N-butyl acetate A₀, HC₀, HD₀ PropanonitrileA₀, HA₀, HC₀, HD₀ Propylene carbonate A₀, HC₀, HD₀ Pyridine S7₀Tert-butanol A₀, HC₀, HD₀ Tetrahydrofuran A₀, HC₀, HD₀ TetrahydropyranHC₀, HD₀ Toluene A₀, HC₀, HD₀ Triethylamine HC₀, HD₀ Water A₀, HA₀, HC₀,HD₀ Xylene A₀, HA₀, HC₀, HD₀

The polymorph screening of Compound I yielded fourteen forms and a newform (Form B₀) obtained only on heating the hydrates above 120° C. Asummary of the results of the isolated forms is shown in Table 16 below.

TABLE 16 Characterization data for isolated forms of Compound I ChemicalPhysical Stability TGA Stability (HPLC (weight (XRPD (post 4 loss post 4weeks at DVS % 25° C. weeks at 40° C./75% increase in XRPD to 40° C./75%RH (Area mass at post DVS Purity Form XRPD DSC 150° C.) RH) %) 90% RHanalysis (%) A₀ Crystalline Melt 0.07%  No 99.0 0.1 No 99.2 endothermsignificant significant at 239.7° C. changes changes B₀ Crystalline Melt— — — — — — endotherm at 199.8° C. HA₀ Crystalline Broad 3.9% No 99.01.5 No 99.6 endotherm significant significant at 99° C. changes changesdue to water loss. Conversion to form B₀ occurs on losing water HC₀Crystalline Broad 3.8% No 99.0  0.44 No 99.6 endotherm significantsignificant at 112° C. changes changes due to water loss. Conversion toform B₀ occurs on losing water HD₀ Crystalline Broad 4.0% No 92.3Insufficient No 93.5 endotherm significant material significant at 110°C. changes changes due to water loss. Conversion to form B0 occurs onlosing waterDescription of Stable Solid State FormsPreparation of Anhydrous Form A₀

Approximately 200 mg of Compound I was slurried in 45 volumes of heptaneat 85° C. for 45 hours, cooled to 65° C. and filter-dried under highvacuum at room temperature for 3 hours. The recovery of Form A₀ was 97%.

In an alternative procedure, the conversion of Compound Ito Form A₀ wasachieved according to the following process:

-   -   1) Compound I was dissolved in 30 volumes of THF. The solution        may be treated with a metal scavenger or carbon at this point,        if desired.    -   2) The resulting solution was filtered to remove the metal        scavenger or carbon followed by a polish filtration through a        1-micron inline cartridge filter to remove any external        particulates.    -   3) The solvent (THF) was partially distilled to approximately        60% of the original volume under vacuum at ambient temperature        followed by the slow addition of an equivalent volume of an        anti-solvent (heptane) to precipitate Compound I.    -   4) Vacuum distillation and addition of more heptane was        continued until the solvent contained less than 5% of THF by        volume.    -   5) The resulting slurry was heated to 90-96° C. and agitated at        this temperature for 3-5 hours to achieve a complete conversion        to Form A₀.    -   6) The slurry was cooled to ambient temperature (25±5° C.).    -   7) The product/Compound I was collected via filtration under a        dry, inert gas to avoid moisture being sucked through the        product.    -   8) The wet cake was dried at up to 80° C. until the residual        solvents in the product met the specifications. Drying may be        performed at atmospheric pressure or under vacuum.        Characterization of Form A₀ Using Variable Temperature X-Ray        Powder Diffraction (VT-XRPD)

No solid-solid transformation takes place in the temperature range 20°C. to 250° C. for Form A₀. After exposure to ambient conditions, thereis no significant change in the XRPD pattern of the sample obtained byheating to 220° C. (See FIG. 15).

Characterization of Form A₀ by Thermal Gravitmetric Analysis (TGA)

Form A₀ shows a single peak at ca. 239° C. with an enthalpy of fusion(ΔH_(Fus)) of 84.4 J/g. No loss of mass is detected by TGA. Theexistence of a desolvation process was discounted because no loss ofweight was detected by TGA (See FIG. 16).

Characterization of Form A₀ by Water Sorption (DVS)

Regular DVS (0 to 90% RH)

The amount of moisture adsorbed at 75% RH was less than 0.08% andapproximately 0.1% at 90% RH. The adsorption and desorption curvesoverlap suggesting that Form A₀ is not hygroscopic (See FIG. 17 andTable 25). No significant changes were observed by XRPD re-analysisafter DVS (FIG. 18).

TABLE 17 DVS Data for Form A₀ (Regular) Form At 75% RH uptake Totaluptake at 90% RH A₀ 0.08 0.1Irregular DVS (90 to 0% RH)

The sample mass only increases at 0.5% at 90% RH. The hysteresis gapsuggests only surface water adsorption is occurring. The isotherm isreversible with a total increase in mass <0.6%. (FIG. 19 and Table 26).No significant changes were observed by XRPD re-analysis after DVS (FIG.18).

TABLE 18 DVS data for Form A₀ (Irregular) Form At 75% RH uptake Totaluptake at 90% RH A₀ 0.15 0.5Characterization of Form A₀ by Fourier Transform Infrared Spectroscopy(FTIR) and Raman Spectroscopy

The FTIR and Raman spectra of the crystalline Form A₀ are shown in FIG.20 and FIG. 21, respectively.

Preparation of Anhydrous Form B₀

Form B₀ was obtained by heating 20 mg of Compound Ito 125° C. undernitrogen flow.

Characterization of Form B₀ by Variable Temperature X-Ray PowderDiffraction (VT-XRPD)

After the dehydration, no solid-solid transformation takes place in therange 150° C. to 200° C. for Form B₀ (See FIG. 22).

Characterization of Form B₀ by Thermal Analysis

The Differential Scanning calorimetry (DSC) diagram of Form B₀ presentsmelting at ca. 197° C. with an enthalpy of fusion (ΔH_(fus)) of 68.2 J/g(FIG. 23). A solid-solid transition occurs before the melting point ofthe form Compound I-B₀. Form B₀ was obtained only by desolvation. Therelative thermodynamic stability of the forms is reflected in the DSCdata shown between 120° C. and 199° C.

Preparation of Hydrate Form HA₀

Crystallization from THF/Heptane

Form HA₀ was obtained as 200 mg of Compound I was precipitated from 70volumes of THF with 143 volumes of heptane at room temperature. Thesolid was isolated by filtration. The material was dried at 57° C. for18 hours.

Preparation by Solid-Solid Transition

Form HA₀ was obtained as 20 mg Compound I was heated to 125° C. andcooled to room temperature without nitrogen flow.

Characterization of Form HA₀ by Thermal Analysis

The DSC thermograms of Form HA₀ show the presence of two differentendothermic peaks (FIG. 24 and Table 27). In an open pan, hydratesexhibit a broad endothermic peak between approximately 60 and 120° C.corresponding to the total amount of water escaping from the crystal.The endothermic event corresponds to the dehydration process involvingthe escape of water from the lattice. Desolvation occurs in the solidstate with an endothermic peak. The position and energy of thisendothermic peak depend on the phase diagram of two components, the drugsubstance and the solvent and the stability of the component formed. TheDSC thermograms of the solvates present broad endothermic peaks attemperatures near the boiling points of their respective solvents thatcan be assigned to desolvation processes, is confirmed by TGA. Themonohydrate Form HA₀, when studied by TGA, demonstrated an averageweight loss of 4.0% between 50 and 120° C. This agrees with thetheoretical value for incorporation of one mole of water with one moleof Compound I is 4.1%

TABLE 19 DSC onset and peak temperatures of desolvation for Form HA₀Principal Peak Solvate Weight onset Temp./ Temp./ Classification Solventloss (%) ° C. ° C. HA₀ Water 4.0 61.6 99.1Characterization of Form HA₀ by Water Sorption

FIG. 25 displays the dynamic vapor sorption data collected on Form HA₀.Upon drying, there is an immediate uptake upon exposure to moisture. Theisotherm of the Form HA₀ shows a 1.25% weight decrease between 20-30%RH. From 30-90% RH the uptake begins to reach equilibrium. During thefirst desorption phase there is a slight hysteresis suggesting onlysurface adsorption. There is almost no desorption during seconddesorption phase, but the sample experiences a second change of ˜0.4%.The sorption shows evidence that this form is a channel hydrate. Thenon-stoichiometric hydration comes from incomplete hydration of thelattice channels. No significant changes were observed on XRPDre-analysis after DVS.

Characterization by FTIR and Raman Spectroscopy

The FTIR and Raman spectra of the crystalline Form HA₀ are shown in FIG.26 and FIG. 27, respectively.

Preparation of Hydrate Form HC₀

Recrystallization from Ethanol/Water

Form HC₀ was obtained as 40 mg of Lot 7 was added in 400 μL of ethanoland 100 μL of water. The sample was heated to an initial temperature of80° C. at a rate of 4.8° C./min and, after 30 minutes, cooled at 0.28°C./min to a final temperature of 5° C. and kept at that temperature for18 hours. The solid was isolated by filtration. The material was driedat 57° C. for 10 hours.

Storage at 40° C./75% RH with the Ethanol Solvate

Form HC₀ was obtained as 20 mg of ethanol solvate of Compound I wasstored at 40° C./75% RH for 9 days.

Preparation of Crystal Structure

Single crystals were prepared by adding 200 mg of Lot 7 solid materialto tetrahydrofuran for the monohydrate HC₀ to assure saturatedconditions at the boiling point. The mixture was cooled and filteredthrough a 0.22μ nylon membrane filter into a warmed glass vial. Thesolution was cooled to 20° C.±0.2° C. in order to increase thesupersaturation value, and the homogeneous solution was left standingfor several days.

Crystal Structure Determination by Single Crystal X-Ray Diffraction

Single crystal X-Ray data was obtained for HC₀ Cell parameters obtainedfrom the data are presented in Table 28.

The data were collected at a temperature of 103K using the ω-2θ scantechnique. A colorless plate of C₂₄H₂₈N₄O₄ having approximate dimensionsof 0.30×0.16×0.11 mm was mounted on a glass fiber in a randomorientation. The triclinic cell parameters (P-1, Z=2) and calculatedvolumes are:

a = 7.6128(10) α = 65.839(18)° b = 11.5697(15) β = 79.137(16)° c =13.193(4)Å γ = 86.800(10)° V = 1040.9(3)Å³.

TABLE 20 Crystal X-ray data collection and refinement parameters forForm HC₀ Identification code Form HC₀ Empirical formula C₂₄H₂₈N₄O₄Formula weight 436.50 Temperature 103(2) K Wavelength, Å 0.71073 Crystalsystem, Space group Triclinic, P-1 Unit cell dimensions a, Å 7.6128(10)b, Å 11.5697(15) c, Å 13.193(4) α, ° 65.839(18) β, ° 79.137(16) γ, °86.800(10) Volume 1040.9(3) Z 2 F(000) 464 Density (calculated), Mg/m31.393 Absorption coefficient, mm−1 0.096 Crystal size, mm3 0.30 × 0.16 ×0.11 Theta range for data collection 3.86 to 28.75°. Index ranges −9 <=h <= 9 −15 <= k <= 15 −16 <= l <= 17 Reflections collected 8739Independent reflections 4527 0.026 Completeness to theta = 28.75° 83.6%Absorption correction None Max. and min. transmission 0.9895 and 0.9716Refinement method Full-matrix least-squares on F2Data/restraints/parameters 4527/0/298 Goodness-of-fit on F2 1.069 FinalR indices [I > 2sigma(I)] R1 = 0.044 wR2 = 0.099 R indices (all data) R1= 0.072 wR2 = 0.112 Largest diff. peak and hole, e.Å-3 0.25 and −0.24Characterization of Form HC₀ by Thermal Analysis

The DSC thermogram of Form HC₀ shows the presence of two differentendothermic peaks (FIG. 28 and Table 29). The monohydrate HC₀, whensubjected to TGA, demonstrated an average weight loss of 3.9% between 50and 120° C. This corresponds to the theoretical value for incorporationof one mole of water with one mole of Compound I of 4.1%.

TABLE 21 DSC onset and peak desolvation temperatures for Form HC₀Principal Peak Solvate Weight onset Temp./ Temp./ Classification Solventloss (%) ° C. ° C. HC₀ Water 3.9 85.9 112.2Characterization of Form HC₀ by Water SorptionRegular DVS (0 to 90% RH)

The hysteresis gap suggests only surface water adsorption is occurringwith a total uptake of 0.4% (FIG. 29 and Table 30). No significantchanges were observed by XRPD re-analysis after DVS (FIG. 30).

TABLE 22 DVS data for Form HC₀ (Regular) Form At 75% RH uptake Totaluptake at 90% RH HC₀ 0.3 0.4Irregular DVS (90 to 0% RH)

The hysteresis gap suggests only surface water adsorption is occurringfrom 0-40% RH. From 40-90% RH there appears to be bulk absorptionoccurring (FIG. 31 and Table 31). No significant changes were observedby XRPD re-analysis after DVS (FIG. 30).

TABLE 23 DVS Data for Form HC₀ (Irregular) Form At 75% RH uptake Totaluptake at 90% RH HC₀ 0.3 0.4Characterization by FTIR and Raman Spectroscopy

The FTIR and Raman spectra of the crystalline Form HC₀ are shown in FIG.32 and FIG. 33, respectively.

Preparation of Hydrate Form HD₀

Recrystallization from Acetone/Water

Form HD₀ was obtained as 40 mg of Lot 7 was added in 400 μL of acetoneand 100 μL of water. The sample was heated to an initial temperature of80° C. at a rate of 4.8° C./min and, after 30 minutes, cooled at 0.28°C./min to a final temperature of 5° C. and kept at that temperature for18 h. The solid was isolated by filtration. The material was dried at57° C. for 10 hours.

Recrystallization from 2-Methyl-2-Propanol

Form HD₀ was obtained as 0.54 g of Compound I in 55 mL of2-methyl-2-propanol was almost completely dissolved by heating to theboiling point. The cloudy solution was syringe filtered using a 5μ nylonmembrane syringe filter to give a clear solution (about 15% spilled andlost). The solution was concentrated to 25-30 mL and chilled for 4.5-5hours at 2-8° C. to give a solid. The solid was melted in the oven at50° C. and insoluble material isolated by suction filtration on a warmapparatus to prevent freezing of t-butyl alcohol. The solid thatresulted was dried in a 50° C. oven for 2 hours to yield 0.42 g (75%recovery).

Recrystallization from Isopropyl Acetate

Form HD₀ was obtained as 0.45 g of Compound I in 7.5 mL of isopropylacetate was stirred for 20 hours at room temperature with a magneticstirring bar in a glass 20 mL scintillation vial with the cap fastenedlightly. The slurry was suction filtered and the solid was allowed todry over 110 hours exposed to air in the fume hood. The dried materialweighed 380 mg (84% recovery).

Preparation of Crystal Structure

Single crystals were prepared by adding 200 mg of Lot 7 solid materialto tetrahydrofuran for the monohydrate HC₀ to assure saturatedconditions at the boiling point. The mixture was cooled and filteredthrough a 0.22μ nylon membrane filter into a warmed glass vial. Thesolution was cooled to 20° C.±0.2° C. in order to increase thesupersaturation value and the homogeneous solution was left standing forseveral days.

Crystal Structure Determination by Single Crystal X-Ray Diffraction

Single crystal X-Ray data was obtained for HD₀. Cell parameters obtainedfrom the data is presented in Table 32 below. The data were collected ata temperature of 103K using the ω-2θ scan technique. A colorless plateof C₂₄H₂₈N₄O₄ having approximate dimensions of 0.40×0.25×0.08 mm wasmounted on a glass fiber in a random orientation. The triclinic cellparameters (P−1, Z=2) and calculated volume are:

a = 8.171(2) α = 111.173(18)° b = 11.419(3) β = 92.863(17)° c =12.7305(19)Å γ = 102.07(2)° V = 1072.8(4)Å^(3.).

TABLE 24 Crystal X-Ray Data Collection and Refinement Parameters for HD₀Identification code Form HD₀ Empirical formula C₂₄H₂₈N₄O₄ Formula weight436.50 Temperature 103(2) K Wavelength, Å 0.71073 Crystal system, Spacegroup Triclinic, P-1 Unit cell dimensions a, Å 8.171(2) b, Å 11.419(3)c, Å 12.7305(19) α, ° 111.173(18) β, ° 92.863(17) γ, ° 102.07(2) Volume1072.8(4) Z 2 F(000) 464 Density (calculated), Mg/m3 1.351 Absorptioncoefficient, mm−1 0.094 Crystal size, mm3 0.40 × 0.25 × 0.08 Theta rangefor data collection 3.95 to 26.56°. Index ranges −10 <= h <= 10 −14 <= k<= 14 −15 <= l <= 15 Reflections collected 9366 Independent reflections4373 0.040 Completeness to theta = 28.75° 97.7% Absorption correctionNone Max. and min. transmission 0.9926 and 0.9635 Refinement methodFull-matrix least-squares on F2 Data/restraints/parameters 4373/0/302Goodness-of-fit on F2 1.166 Final R indices [I > 2sigma(I)] 0.051 0.099R indices (all data) 0.087 0.113 Largest diff. peak and hole, e.Å-3 0.24and −0.25Characterization of Form HD₀ by Thermal Analysis

The DSC thermograms of Form HD₀ show the presence of two differentendothermic peaks (FIG. 34 and Table 33). The monohydrate HD₀, whensubjected to TGA, demonstrated an average weight loss of 4.0% between 50and 120° C. The theoretical value for incorporation of one mole of waterwith one mole of Compound I is 4.1%.

TABLE 25 Onset and DSC Peak of desolvation temperatures of Form HD₀Principal Peak Solvate Weight onset Temp./ Temp./ Classification Solventloss (%) ° C. ° C. HD₀ Water 4.0 86.3 110.7Characterization of Form HD₀ by Water SorptionRegular DVS (0 to 90% RH)

The sample mass only increases 0.6% at 90% RH. The hysteresis gapsuggests that surface water adsorption and bulk absorption is occurring(FIG. 35 and Table 34). No significant changes were observed by XRPDre-analysis after DVS (FIG. 36).

TABLE 26 DVS data for form HD₀ (regular) Form At 75% RH uptake Totaluptake at 90% RH HD₀ 0.4 0.6Irregular DVS (90 to 0% RH)

The sample mass only increases 0.8% at 90% RH. The hysteresis gapsuggests that surface water adsorption and limited bulk absorption isoccurring (FIG. 37 and Table 26). No significant changes were observedby XRPD re-analysis after DVS (FIG. 27).

TABLE 27 DVS data for form HD₀ (irregular) Form At 75% RH uptake Totaluptake at 90% RH HD₀ 0.4 0.8Characterization of Form HD₀ by FTIR and Raman Spectrometry

The FTIR and Raman spectra of the crystalline Form HD₀ are shown in FIG.38 and FIG. 39, respectively.

Solvate Forms of Compound I

Recrystallization from Methanol

Form S2₀ was obtained as 40 mg of Lot 7 was added in 400 μL of methanol.The sample was slurried at 20° C.±0.2 for 3 days. The solid was isolatedby filtration. The material was dried at 57° C. for 10 hours.

Recrystallization from 2-Propanol

Form S3₀ was obtained as 40 mg of Lot 7 was added in 400 μL of2-propanol. The sample was slurried at 20° C.±0.2 for 3 days. The solidwas isolated by filtration. The material was dried at 57° C. for 10hours.

Recrystallization from Ethanol

Form S4₀ was obtained as 40 mg of Lot 7 was added in 400 μL of ethanol.The sample was slurried at 20° C.±0.2 for 3 days. The solid was isolatedby filtration. The material was dried at 57° C. for 10 hours.

Preparation of Crystal Structure

Single crystals were prepared by adding 200 mg of Lot 7 solid materialto ethanol for the ethanolate to assure saturated conditions at theboiling point. The mixture was cooled and filtered through a 0.22 μmnylon membrane filter into a warmed glass vial. The solution was cooledto 20° C.±0.2° C. in order to increase the supersaturation value and thehomogeneous solution was left standing for several days.

Recrystallization from N—N-dimethylformamide

Form S5₀ was obtained as 40 mg of Lot 7 was added in 400 μL ofN—N-dimethylformamide (DMF). The sample was slurried at 20° C.±0.2 for 3days. The solid was isolated by filtration. The material was dried at57° C. for 10 hours.

Recrystallization from Ethylene Glycol

Form S6₀ was obtained as 40 mg of Lot 7 was added in 400 μL of ethyleneglycol. The sample was heated to an initial temperature of 80° C. at arate of 4.8° C./min and, after 30 minutes, cooled at 0.28° C./min to afinal temperature of 5° C. and kept at that temperature for 18 hours.The solid was isolated by filtration. The material was dried at 57° C.for 10 hours.

Recrystallization from Pyridine

Form S7₀ was obtained as 40 mg of Lot 7 was added in 400 μL of pyridine.The sample was heated to an initial temperature of 80° C. at a rate of4.8° C./min and, after 30 minutes, cooled at 0.28° C./min to a finaltemperature of 5° C. and kept at that temperature for 18 hours. Thesolid was isolated by filtration. The material was dried at 57° C. for10 hours.

Recrystallization from 1-propanol

Form S9₀ was obtained as 40 mg of Compound I in 1-propanol to assuresaturated conditions at the boiling point. The mixture was cooled andfiltered through a 5μ nylon membrane filter into a warmed glass vial.The solution was cooled to RT and placed in a refrigerator (ca. 4° C.)until crystal formation appeared to reach completion as determined byvisual inspection. Samples difficult to decant were centrifuged at 12000rpm for four minutes.

Recrystallization from N—N-dimethylacetamide

Form S10₀ was obtained as 40 mg of Lot 7 was added in 400 μL ofN—N-dimethylacetamide (DMA). The sample was slurried at 20° C.±0.2 for 3days. The solid was isolated by filtration. The material was dried at57° C. for 10 hours.

Recrystallization from Isobutanol

Form S12₀ was obtained as 40 mg of Compound I in isobutanol to assuresaturated conditions at the boiling point. The mixture was cooled andfiltered through a 5μ nylon membrane filter into a warmed glass vial.The solution was cooled to RT and placed in a refrigerator (ca. 4° C.)until crystal formation appeared to reach completion as determined byvisual inspection. Samples difficult to decant were centrifuged at 12000rpm for four minutes.

Crystal Structure Determination by Single Crystal X-Ray Diffraction

Single crystal X-Ray data were obtained for Form S4₀. Cell parametersobtained from the data are presented in Table 28.

TABLE 28 Crystal X-ray data collection and refinement parameters forethanol solvate S4₀ From Single Crystal After Rietveld on S4₀ Unit celldimensions a, Å 8.828(3) 8.996(7) b, Å 11.652(3) 11.813(2) c, Å13.234(6) 13.191(9) α, ° 115.01(3) 114.28(1) β, ° 108.09(3) 108.52(8) γ,° 93.00(2) 92.56(0)Thermal Analysis of the Solvate Forms of Compound I

DSC thermal curves showed the presence of a large and broad endothermbefore the melting point of Compound I for all the solvates. TGA studiesshowed that these endotherms can be attributed to a desolvation processfor Forms S2₀ (FIG. 40), S3₀ (FIG. 41), S4₀ (FIG. 42), S5₀ (FIG. 43),S6₀ (FIG. 44), S7₀ (FIG. 45), S9₀ (FIG. 46), S10₀(FIG. 47), and S12₀(FIG. 48). The calculation of the solvent weight loss led for thesesolvents is presented in Table 29.

TABLE 29 DSC onset and peak desolvation temperatures for solvate formsSolvate Solvate Principal Weight loss Weight loss onset Peak (%) (%)Temp./ Temp./ Form Solvent Experimental Theory 1:1 ° C. ° C. S2₀Methanol 7.3 7.1 87.5 111.8 S3₀ 2-propanol 10.41 12.5 78.0 104.9 S4₀Ethanol 8.7 9.9 109.9 122.8 S5₀ DMF 13.0 14.9 127.0 147.0 S6₀ Ethyleneglycol 6.16 12.9 139.9 156.6 S7₀ Pyridine 3.53 15.9 128.0 141.9 S9₀1-propanol 12.42 12.5 118.4 129.6 S10₀ DMA 19.42 17.2 135.0 156.0 S12₀Isobutanol 11.54 15.0 81.8 132.5Crystal Structure Determination of Compound I Monohydrates

The hydrate Forms HC₀ and HD₀ are isostructural, which means that theindividual ‘isomorphic solvates’ crystallize in the same space groupwith only small distortions of the unit cell dimensions and the sametype of molecular network of the host molecules (Reutzel-Edens S. M.,Newman A W., Polymorphism in the Pharmaceutical Industry, Edited by RolfHilfiker, 2006, Wiley-VCH Verlag GmbH & Co. KGaA ISBN: 9783527311460).Form HC₀ and Form HD₀ differ in the conformation of thetetrahydropyrazine ring. The X-ray powder diffraction pattern of the twohydrate forms could be successfully refined using Rietveld techniques(Rietveld H. M. “A profile refinement method for nuclear and magneticstructures”, Journal of Applied Crystallography 2: 65-71 (1969)) withthe single crystal parameters as the starting point. Details of the celldata, data collection and refinement are summarized in Table 28 andTable 32.

FTIR and FT-Raman Method for Identification Assay

Comparison of the FTIR and Raman spectra in FIG. 20 and FIG. 21 for FormA₀ and for the hydrates (FIG. 26, FIG. 32, FIG. 38) and (FIG. 27, FIG.33, FIG. 39) show little difference except in the carbonyl stretchingregion for the FTIR.

In A₀, a peak occurred at 1765 cm⁻¹ of medium intensity for the FTIR and1770 cm⁻¹ for the Raman. In the hydrate forms, a peak occurred in thisregion at 1742 cm⁻¹ of medium intensity for FTIR and 1754-1695 cm⁻¹ forthe Raman. This absorption peak is assigned to the imide carbonylfunctionality contained within the five-membered ring of the Compound Istructure. This difference is large enough that it could be used foridentification of nearly pure solid state forms. IR spectra for hydrateforms and A₀ show some differences but the most significant concerns thebroad band (3800-2800) present in hydrate forms due to the stretching ofthe —OH bond in the hydroxyl group.

TABLE 30 Frequencies (cm⁻¹) and attribution of fundamental vibration forCompound I for FTIR Form -Hydroxyl (cm⁻¹) -Carbonyl (cm⁻¹) A₀ 3349.91765.3 HA₀ 3498.2 1742.0 HC₀ 3498.2 1742.0 HD₀ 3498.2 1742.0

TABLE 31 Frequencies (cm⁻¹) and attribution of fundamental vibration forCompound I for Raman Form -Carbonyl (cm⁻¹) -Carbonyl (cm⁻¹) A₀ 1770 1638HA₀ 1754 1699 HC₀ 1752 1696 HD₀ 1748 1695Relationship between Solid State Forms

Relative Stability of Slurries of Compound I in Water

When hydrate HA₀ and A₀ are crystallized from aqueous media, a mixtureof Forms HC₀+HD₀ is produced (Table 32).

TABLE 32 Crystal forms obtained per well plate of Compound I Form of theVolumes of Starting Material Solvent Water Forms Found HA₀ 0.175 HC₀,HD₀ A₀ Methyl acetate 0 A₀ A₀ Methyl acetate 0.175 HC₀, HD₀ A₀ Methylacetate 0.25 HC₀, HD₀ A₀ Methyl acetate 1 HC₀, HD₀ A₀ Methyl acetate 1.5HC₀, HD₀

Two ml of water was added to a few milligrams of Compound I forms. Thesamples were slurried overnight. A small sample of solid was removed andanalyzed by XRPD. After slurrying, Forms HA₀ and A₀ were found to haveconverted to the hydrate forms (mixture of Forms HC₀ and HD₀) under allof the conditions investigated (Table 33, Table 34, Table 35, Table 36and Table 37). The hydrate form appears to be more thermodynamicallystable than Form A₀ between 5 and 45° C.

TABLE 33 XRPD analysis of residual solid from thermodynamic solubilityexperiments of Form A₀ in water at 5° C. Form A₀/mg Time (days) FormsFound 15.9 1 HC₀, HD₀ 14.1 4 HC₀, HD₀

TABLE 34 XRPD analysis of residual solid from thermodynamic solubilityexperiments of Form HA₀ and Form HB₀ (mixture of HC₀ and HD₀) in waterat RT HB₀/mg HA₀/mg Time (days) Forms Found 5.05 6.08 1 HA₀, HC₀, HD₀4.91 4.98 9 HC₀, HD₀ 4.94 5.02 10 HC₀, HD₀, trace of HA₀ 5.34 5.57 14HC₀, HD₀, trace of HA₀

TABLE 35 XRPD analysis of residual solid from thermodynamic solubilityexperiments of Form HA₀ and Form HC₀ in water at RT HC₀/mg HA₀/mg Time(days) Forms Found 5.34 5.35 1 HA₀, HC₀ 5.23 6.04 2 HA₀, HD₀ 5.19 5.21 5HC₀, HD₀

TABLE 36 XRPD analysis of residual solid from thermodynamic solubilityexperiments Form HA₀ and Form HD₀ in water at RT HD₀/mg HA₀/mg Time (days) Forms Found 5.48 5.83 1 HA₀, HD₀ 5.43 5.06 2 HA₀, HC₀ 5.10 5.18 5HD₀, HC₀ 5.41 6.01 14 HD₀, HC₀

TABLE 37 XRPD analysis of residual solid from thermodynamic solubilityexperiments Form A₀ at 45° C. in water Form A₀/mg Time (days) FormsFound 14.3 1 HA₀, HC₀Relative Stability of Monohydrates

By measuring the thermodynamic solubility of two polymorphs (Form HC₀and Form HD₀) at a practical range of temperatures, it is possible todetermine which is the more stable and whether the relationship betweenthem is monotropic or enantiotropic. Experiments were set up to measurethe thermodynamic solubility of these monohydrate forms at roomtemperature and 55° C. in ethyl acetate, MTBE and 1-pentanol. Thesesolvents were selected as Compound I did not form solvates in thesesolvents during the polymorph screen.

TABLE 38 XRPD analysis of residual solid from thermodynamic solubilityexperiments of monohydrate forms Solu- Temper- XRPD Analysis XRPDAnalysis bility Solvent ature after 1 days after 3 days (mg/mL) Ethyl RTHD₀ > HC₀ HD₀ 1.7 acetate Ethyl 55° C. HD₀ HD₀ > HC₀ 2.6 acetate MTBE RTHD₀, HC₀ HD₀, HC₀ 1.8 MTBE 55° C. HD₀ and HD₀, HC₀ 1.9 Form HC₀1-pentanol RT HD₀ > HC₀ HD₀ > HC₀ 6.8 1-pentanol 55° C. HD₀ > HC₀ HD₀ >HC₀ 28.7

Results summarized in Table 38 show that for the solvents used and thetemperature ranges explored, the solubility values of the two hydratedpolymorphs are very close but always higher for Form HD₀. It doesindicate that between room temperature and 55° C. in solution Form HD₀is more thermodynamically stable than Form HC₀ and form HA₀.

Solid State Stress Stability

Stress stability studies were performed to get a timely impression ofthe influence of temperature and humidity on form stability. Astability-indicating HPLC assay method was developed for quantitation ofCompound I and its major degradation product,7-methoxy-1,2,3,11-tetrahydro-5,11-diaza-benzo[a]trindene-4,6-dione,previously referred to as “Compound E”. The developed method isspecific, accurate, precise and robust. The procedure permitted anaccurate and quantitative determination of Compound I and Compound E.All the degradation products formed during forced decomposition studieswere well separated from the main peaks demonstrating that the developedmethod was specific and stability-indicating.

Form A₀

In the solid state, anhydrous Form A₀ showed a tendency to take up waterfrom the environment and to give rise under standard ICH stressedconditions, 40° C. and 75% RH to hydrate Forms HC₀ and HD₀ after 3months. Chemical degradation was not observed in Compound I samplesunder these stressed conditions. Chemical degradation was only observedwhen Compound I was exposed to 110° C. (Table 39, Table 40 and Table41).

TABLE 39 Stability of Form A₀ at 40° C./75% RH SAMPLE Elapsed TimeNUMBER (Days) XRPD DSC HPLC 1 5 A₀ 239.7 99.2% Compound I, 0.8% CompoundE 2 16 A₀ 239.4 99.2% Compound I, 0.8% Compound E 3 29 A₀ 239.5 99.2%Compound I, 0.8% Compound E 4 141 A₀, HC₀, HD₀ 240.0 99.0% Compound I,0.9% Compound E

TABLE 40 Stability of Form A₀ at 60° C./0% RH SAMPLE Elapsed Time NUMBER(Days) XRPD DSC HPLC 1 7 A₀ 236.2 99.3% Compound I, 0.6% Compound E 2 14A₀ 236.1 99.1% Compound I, 0.8% Compound E 3 28 A₀ 236.4 99.1% CompoundI, 0.5% Compound E

TABLE 41 Stability of Form A₀ at 110° C. SAMPLE Elapsed Time NUMBER(Days) XRPD DSC HPLC 1 7 A₀ 236.2 98.7% Compound I, 1.1% Compound E 2 14A₀ 235.6 95.6% Compound I, 4.4% Compound E 3 28 A₀ 238.0 93.4% CompoundI, 6.2% Compound EMonohydrate Forms

In the solid state, Table 42, Table 43 and Table 44 show that allcrystalline monohydrates were stable for 28 days when stored at 40° C.and 75% relative humidity.

TABLE 42 Stability of Form HA₀ at 40° C./75% RH SAMPLE Elapsed TimeNUMBER (Days) XRPD HPLC 1 0 HA₀ 99.6% Compound I, ND Compound E 2 7 HA₀99.5% Compound I, 0.1% Compound E 3 26 HA₀ 99.0% Compound I, 0.4%Compound E ND = non-detectable

TABLE 43 Stability data for Form HC₀ at 40° C./75% RH SAMPLE ElapsedTime NUMBER (Days) XRPD HPLC 1 0 HC₀ 92.6% Compound I, 0.3% Compound E 228 HC₀ 93.3% Compound I, 0.5% Compound E, 0.2% others

TABLE 44 Stability data for Form HD₀ at 40° C./75% RH SAMPLE ElapsedTime NUMBER (Days) XRPD HPLC 1 0 HD₀ 93.5% Compound I, 0.3% Compound E 27 HD₀ 92.2% Compound I, 0.9% Compound E 3 13 HD₀ 92.3% Compound I, 0.7%Compound E 4 28 HD₀ 92.3% Compound I, 0.7% Compound EForm S4₀

Form S4₀, an ethanol solvate, is transformed into monohydrate Form HC₀after 9 days at 40° C./75% RH and remained in this state for 62 days(Table 45).

TABLE 45 Stability data for S4₀ at 40° C./75% RH SAMPLE Elapsed TimeNUMBER (Days) XRPD HPLC 1 9 HC₀ 99.4% Compound I, 0.35% Compound E 2 17HC₀ 99.4% Compound I, 0.36% Compound E 3 31 HC₀ 99.4% Compound I, 0.47%Compound E 4 62 HC₀ 99.2% Compound I, 0.47% Compound EForm Conversion Mechanical StressGrinding by Mortar and Pestle

Approximately 100 mg of Compound I was ground at different times rangingfrom 5 to 27 min in an agate mortar. Samples were removed for XRPD andthermal analysis. The grinding process was stopped every 5 minutes toscrape and remix powder cakes at the curvature end of the jars to ensurehomogenous grinding.

Milling by Wig-L-Bug®

A Wig-l-Bug® (Piketech, USA) was used to grind Compound I Form A₀, HA₀and HB₀ (mixture of Forms HC₀ and HD₀). Each sample (50 mg) was groundfor periods of 5 and 10 minutes or until no change was observed. Eachmilling was carried out in a 2.82 cm³ container using 0.9 g stainlesssteel ball (0.6 mm diameter). The vial is swung through a 6.5° arc at3200 rpm, causing the ball to strike the end of the vial at over 100 Hz.

Form A₀Stability

After twenty minutes (mortar and pestle) and five minutes (Wig-l-Bug®),the XRPD patterns showed that crystallinity had been significantlyreduced. As the remaining peaks were in the same position as thestarting material, the samples did not become completely amorphous (FIG.49).

Forms HA₀, HC₀ and HD₀ Stability

After three five-minute grinding intervals, the XRPD pattern for groundForm HB₀ (a mixture of Forms HC₀ and HD₀) (FIG. 50) is similar to thepattern for ground HA₀. The XRPD peak at 7.6° (2θ) is reduced inintensity by a factor of approximately 30.

The DSC curves show a broad endotherm ranging from 50 to 100° C. thatcan be attributed to the release of water. The thermogram shows first aglass transition Tg located at ca. 113° C. (FIG. 51). The DSC indicatesthat the one observed exotherm corresponds to a one steprecrystallization at 136° C. towards the metastable Form B₀. A broadendothermic event, that corresponds to the melting of the B₀ form and afinal melt at 231° C. (Form A₀). An explanation for these events can begiven if Forms A₀ and B₀ are considered to be monotropic, where Form A₀is the more stable form.

It is meant to be understood that peak heights obtained as a result ofthe XRPD, VT-XRPD and single crystal diffraction pattern experiments mayvary and will be dependent on variables such as the temperature, crystalsize or morphology, sample preparation, or sample height in the analysiswell of the PANalytical X Pert Pro diffractometer or Oxford diffractionCCD diffractometer.

It is also meant to be understood that peak positions may vary whenmeasured with different radiation sources. For example, Cu—Kα₁, Mo—Kα,Co—Kα and Fe—Kα radiation, having wavelengths of 1.54060 Å, 0.7107 Å,1.7902 Å and 1.9373 Å, respectively, may provide peak positions thatdiffer from those measured with Cu—Kα radiation.

It is further meant to be understood that the term “±0.2 degrees2-theta” following a series of peak positions means that all of thepeaks of the group which it follows are reported in terms of angularpositions with a variability of ±0.2 degrees 2-theta. For example,“6.81, 8.52, 9.73, 12.04 and/or 13.25±0.2 degrees 2-theta” means“6.81±0.2 degrees 2-theta, 8.52±0.2 degrees 2-theta, 9.73±0.2 degrees2-theta, 12.04±0.2 degrees 2-theta and/or 13.25±0.2 degrees 2-theta”.

As those skilled in the art will appreciate, numerous modifications andvariations of the present invention are possible in view of the aboveteachings. It is therefore understood that within the scope of theappended claims, the invention can be practiced otherwise than asspecifically described herein, and the scope of the invention isintended to encompass all such variations.

Accordingly, a first embodiment of the invention provides a crystallineform of Compound I that is Form A₀ or Form B₀, or a mixture thereof.

A second embodiment of the invention provides the crystalline form ofthe first embodiment, wherein the crystalline form is Form A₀.

A third embodiment of the invention provides the crystalline form of thefirst embodiment, wherein the crystalline form is Form B₀.

A fourth embodiment of the invention provides the crystalline form ofthe second embodiment, characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 4.32, 6.07, 8.55,12.07 and 15.37±0.2 degrees 2-theta.

A fifth embodiment of the invention provides the crystalline form of thesecond embodiment, having an X-ray powder diffraction patternsubstantially as depicted in FIG. 1.

A sixth embodiment of the invention provides the crystalline form of thethird embodiment, characterized by an X-ray powder diffraction patterncomprising one or more of the following peaks: 7.16, 7.89, 10.77, 16.54,and 21.20±0.2 degrees 2-theta.

A seventh embodiment of the invention provides the crystalline form ofthe third embodiment, having an X-ray powder diffraction patternsubstantially as depicted in FIG. 2.

An eighth embodiment of the invention provides a crystalline form ofCompound I that is Form HA₀, Form HC₀ or Form HD₀ or a mixture thereof.

A ninth embodiment of the invention provides the crystalline form of theeighth embodiment, wherein the crystalline form is Form HA₀.

A tenth embodiment of the invention provides the crystalline form of theeighth embodiment, wherein the crystalline form is Form HC₀.

An eleventh embodiment of the invention provides the crystalline form ofthe eighth embodiment, wherein the crystalline form is Form HD₀.

A twelfth embodiment of the invention provides the crystalline form ofthe ninth embodiment, characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 7.59, 15.12,16.06, 17.94 and 23.89±0.2 degrees 2-theta.

A thirteenth embodiment of the invention provides the crystalline formof the ninth embodiment, having an X-ray powder diffraction patternsubstantially as depicted in FIG. 3.

A fourteenth embodiment of the invention provides the crystalline formof the tenth embodiment, characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 8.36, 8.71,16.69, 17.39 and 24.59±0.2 degrees 2-theta.

A fifteenth embodiment of the invention provides the crystalline form ofthe tenth embodiment, having an X-ray powder diffraction patternsubstantially as depicted in FIG. 4.

A sixteenth embodiment of the invention provides the crystalline form ofthe eleventh embodiment, characterized by an X-ray powder diffractionpattern comprising one or more of the following peaks: 7.60, 8.99 and15.16±0.2 degrees 2-theta.

A seventeenth embodiment of the invention provides the crystalline formof the eleventh embodiment, having an X-ray powder diffraction patternsubstantially as depicted in FIG. 5.

An eighteenth embodiment of the invention provides a crystalline form ofCompound I that is Form S2₀, Form S3₀, Form S4₀, Form S5₀, Form S6₀,Form S7₀, Form S9₀, Form S10₀ or Form S12₀ or a mixture thereof.

A nineteenth embodiment of the invention provides the crystalline formof the eighteenth embodiment, wherein the crystalline form is Form S2₀.

A twentieth embodiment of the invention provides the crystalline form ofthe eighteenth embodiment, wherein the crystalline form is Form S3₀.

A twenty-first embodiment of the invention provides the crystalline formof the eighteenth embodiment, wherein the crystalline form is Form S4₀.

A twenty-second embodiment of the invention provides the crystallineform of the eighteenth embodiment, wherein the crystalline form is FormS5₀.

A twenty-third embodiment of the invention provides the crystalline formof the eighteenth embodiment, wherein the crystalline form is Form S6₀.

A twenty-fourth embodiment of the invention provides the crystallineform of the eighteenth embodiment, wherein the crystalline form is FormS7₀.

A twenty-fifth embodiment of the invention provides the crystalline formof the eighteenth embodiment, wherein the crystalline form is Form S9₀.

A twenty-sixth embodiment of the invention provides the crystalline formof the eighteenth embodiment, wherein the crystalline form is Form S10₀.

A twenty-seventh embodiment of the invention provides the crystallineform of the eighteenth embodiment, wherein the crystalline form is FormS12₀.

A twenty-eighth embodiment of the invention provides the crystallineform of the nineteenth embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.56,14.64, 16.07, 22.24 and 23.02±0.2 degrees 2-theta.

A twenty-ninth embodiment of the invention provides the crystalline formof the twentieth embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 6.70,8.67, 13.36, 16.80 and 16.85±0.2 degrees 2-theta.

A thirtieth embodiment of the invention provides the crystalline form ofthe twenty-first embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.42,8.60, 13.92, 17.20 and 24.46±0.2 degrees 2-theta.

A thirty-first embodiment of the invention provides the crystalline formof the twenty-second embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 4.46,7.67, 8.86 and 11.71±0.2 degrees 2-theta.

A thirty-second embodiment of the invention provides the crystallineform of the twenty-third embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.68,11.10, 16.94, 17.39 and 23.31±0.2 degrees 2-theta.

A thirty-third embodiment of the invention provides the crystalline formof the twenty-fourth embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 4.50,7.70, 8.90 and 11.76±0.2 degrees 2-theta.

A thirty-fourth embodiment of the invention provides the crystallineform of the twenty-fifth embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.34,8.67, 16.68, 17.33 and 24.57±0.2 degrees 2-theta.

A thirty-fifth embodiment of the invention provides the crystalline formof the twenty-sixth embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 4.45,7.62, 8.79, 11.62 and/or 17.67±0.2 degrees 2-theta.

A thirty-sixth embodiment of the invention provides the crystalline formof the twenty-seventh embodiment, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 7.63,7.67, 9.00, 17.99 and 24.46±0.2 degrees 2-theta.

A thirty-seventh embodiment of the invention provides a process forpreparing a crystalline form of Compound I that is Form A₀, comprisingthe steps of:

-   -   a. Slurrying Compound I in a hydrocarbon(s) (such as heptane or        toluene);    -   b. Cooling the resulting slurry;    -   c. Filtering the resulting slurry; and    -   d. Drying the filter-cake.

A thirty-eighth embodiment of the invention provides the process of thethirty-seventh embodiment, wherein Compound I is slurried in 26 to 45volumes of heptane.

A thirty-ninth embodiment of the invention provides the process of thethirty-eighth embodiment, wherein Compound I is slurried in 45 volumesof heptane.

A fortieth embodiment of the invention provides the process of thethirty-seventh embodiment, wherein step (a) is performed at 79 to 83° C.

A forty-first embodiment of the invention provides the process of thefortieth embodiment, wherein step (a) is performed at 85° C.

A forty-second embodiment of the invention provides the process of thethirty-seventh embodiment, wherein step (a) is performed for 24 to 48hours.

A forty-third embodiment of the invention provides the process of theforty-second embodiment, wherein step (a) is performed for 45 hours.

A forty-fourth embodiment of the invention provides the process of thethirty-seventh embodiment, wherein step (b) occurs at a temperature of30-65° C.

A forty-fifth embodiment of the invention provides the process of theforty-fourth embodiment, wherein step (b) occurs at a temperature of 65°C.

A forty-sixth embodiment of the invention provides the process of thethirty-seventh embodiment, wherein step (c) is performed at roomtemperature for 0.33 to 3 hours.

A forty-seventh embodiment of the invention provides the process of theforty-sixth embodiment, wherein step (c) is performed at roomtemperature for three hours.

A forty-eighth embodiment of the invention provides a process forpreparing a crystalline form of Compound I that is Form A₀, comprisingthe steps of:

-   -   a. dissolving Compound I in a solvent;    -   b. filtering the resulting solution;    -   c. partially distilling the solvent while adding an anti-solvent        to precipitate Compound I;    -   d. further distilling the resulting slurry while adding        additional anti-solvent to reduce the volume of the solvent used        in step (a);    -   e. heating the slurry to achieve complete conversion to Form A₀;    -   f. cooling;    -   g. collecting the product via filtration; and    -   h. drying.

A forty-ninth embodiment of the invention provides the process of theforty-eighth, wherein step (a) is performed using 27 to 35 volumes ofTHF.

A fiftieth embodiment of the invention provides the process of theforty-ninth embodiment, wherein step (a) is performed using 30 volumesof THF.

A fifty-first embodiment of the invention provides the process of theforty-eighth embodiment, wherein the solution produced via step (a) mayoptionally be treated with a metal scavenger or carbon.

A fifty-second embodiment of the invention provides the process of theforty-eighth embodiment, wherein the filtering step (b) comprises one orboth of the following steps:

-   -   (i) filtering to remove the metal scavenger; and    -   (ii) polish filtering through a 1-micron inline cartridge        filter.

A fifty-third embodiment of the invention provides the process of theforty-eighth, wherein the solvent present in step (c) is distilled to 60to 90% of its original volume.

A fifty-fourth embodiment of the invention provides the process of theforty-eighth embodiment, wherein step (c) is performed using heptane asthe anti-solvent.

A fifty-fifth embodiment of the invention provides the process of theforty-eighth, wherein step (d) is performed until less than 5% THF byvolume remains.

A fifty-sixth embodiment of the invention provides the process of theforty-eighth embodiment, wherein step (e) is performed at a temperatureof about 90 to 96° C.

A fifty-seventh embodiment of the invention provides the process of theforty-eighth embodiment, wherein step (e) may be optionally omitted.

A fifty-eighth embodiment of the invention provides the process of thefifty-sixth embodiment, wherein the slurry is agitated for about 3 to 5hours.

A fifty-ninth embodiment of the invention provides the process of theforty-eighth embodiment, wherein step (f) is performed at ambienttemperature (25±5° C.).

A sixtieth embodiment of the invention provides the process of theforty-eighth embodiment, wherein the filtration of step (g) is performedusing a dry, inert gas.

A sixty-first embodiment of the invention provides the process of theforty-eighth embodiment, wherein step (h) is performed at a temperatureup to 80° C.

A sixty-second embodiment of the invention provides the process of theforty-eighth embodiment, wherein the residual water and/or solvate(s)are azeotropically removed.

A sixty-third embodiment of the invention provides a pharmaceuticalcomposition comprising Form A₀, Form B₀, Form HA₀, Form HC₀, Form HD₀,or a mixture thereof.

A sixty-fourth embodiment of the invention provides a process for thepreparation of Compound I,

comprising the step of reacting Compound A,

with 1,1,1-trifluoromethanesulfonic acid 1-cyclopenten-1-yl ester toproduce Compound B,

A sixty-fifth embodiment of the invention provides a process for thepreparation of Compound I

comprising the step of reacting Compound C,

with maleimide to produce compound D,

We claim:
 1. A crystalline form of Compound I that is Form A₀ or FormB₀, or a mixture thereof.
 2. The crystalline form of claim 1, whereinthe crystalline form is Form A₀.
 3. The crystalline form of claim 1,wherein the crystalline form is Form B₀.
 4. The crystalline form ofclaim 2, characterized by an X-ray powder diffraction pattern comprisingone or more of the following peaks: 4.32, 6.07, 8.55, 12.07 and15.37±0.2 degrees 2-theta.
 5. The crystalline form of claim 2, having anX-ray powder diffraction pattern substantially as depicted in FIG.
 1. 6.The crystalline form of claim 3, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 7.16,7.89, 10.77, 16.54, and 21.20±0.2 degrees 2-theta.
 7. The crystallineform of claim 3, having an X-ray powder diffraction patternsubstantially as depicted in FIG.
 2. 8. A crystalline form of Compound Ithat is Form HA₀, Form HC₀ or Form HD₀ or a mixture thereof.
 9. Thecrystalline form of claim 8, wherein the crystalline form is Form HA₀.10. The crystalline form of claim 8, wherein the crystalline form isForm HC₀.
 11. The crystalline form of claim 8, wherein the crystallineform is Form HD₀.
 12. The crystalline form of claim 9, characterized byan X-ray powder diffraction pattern comprising one or more of thefollowing peaks: 7.59, 15.12, 16.06, 17.94 and 23.89±0.2 degrees2-theta.
 13. The crystalline form of claim 9, having an X-ray powderdiffraction pattern substantially as depicted in FIG.
 3. 14. Thecrystalline form of claim 10, characterized by an X-ray powderdiffraction pattern comprising one or more of the following peaks: 8.36,8.71, 16.69, 17.39 and 24.59±0.2 degrees 2-theta.
 15. The crystallineform of claim 10, having an X-ray powder diffraction patternsubstantially as depicted in FIG.
 4. 16. The crystalline form of claim11, characterized by an X-ray powder diffraction pattern comprising oneor more of the following peaks: 7.60, 8.99 and 15.16±0.2 degrees2-theta.
 17. The crystalline form of claim 11, having an X-ray powderdiffraction pattern substantially as depicted in FIG.
 5. 18. Acrystalline form of Compound I that is Form S2₀, Form S3₀, Form S4₀,Form S5₀, Form S6₀, Form S7₀, Form S9₀, Form S10₀ or Form S12₀ or amixture thereof.
 19. A process for preparing a crystalline form ofCompound I that is Form A₀ according to claim 2, comprising the stepsof: a. Slurrying Compound I in a hydrocarbon(s) (such as heptane ortoluene); b. Cooling the resulting slurry; c. Filtering the resultingslurry; and d. Drying the filter-cake.
 20. A process for preparing acrystalline form of Compound I that is Form A₀ according to claim 2,comprising the steps of: a. dissolving Compound I in a solvent; b.filtering the resulting solution; c. partially distilling the solventwhile adding an anti-solvent to precipitate Compound I; d. furtherdistilling the resulting slurry while adding additional anti-solvent toreduce the volume of the solvent used in step (a); e. heating the slurryto achieve complete conversion to Form A₀; f. cooling; g. collecting theproduct via filtration; and h. drying.
 21. A pharmaceutical compositioncomprising Form A₀, Form B₀, Form HA₀, Form HC₀, Form HD₀, or a mixturethereof.
 22. A pharmaceutical composition comprising Form A₀, Form B₀ ora mixture thereof.